Systems and methods for processing-nucleic acid molecules from a single cell using sequential co-partitioning and composite barcodes

ABSTRACT

Provided herein are methods, compositions, and systems for multiplexed analysis of individual cells or cell populations. Cells encapsulated in beads and/or biomolecules are sequentially co-partitioned, allowing for analysis of two different types of biomolecules (e.g., RNA and DNA). The present invention leverages different polymer dissociation mechanisms, accompanied with barcoding of biomolecules (e.g., nucleic acid molecules) for multiplexed measurements in single cells. Sequential co-partitioning and barcode technology enables identification and quantitation of DNA and RNA from single cells.

CROSS-REFERENCE

This application is a continuation of U.S. Application Pat. No. 16/708,214, filed Dec. 9, 2019, which claims the benefit of U.S. ProvisionalPat. Application No. 62/777,402, filed Dec. 10, 2018, which areincorporated by reference herein in their entirety for all purposes.

BACKGROUND

A sample may be processed for various purposes, such as identificationof a type of moiety within the sample. The sample may be a biologicalsample. Biological samples may be processed, such as for detection of adisease (e.g., cancer) or identification of a particular species. Thereare various approaches for processing samples, such as polymerase chainreaction (PCR) and sequencing.

Biological samples may be processed within various reactionenvironments, such as partitions. Partitions may be wells or droplets.Droplets or wells may be employed to process biological samples in amanner that enables the biological samples to be partitioned andprocessed separately. For example, such droplets may be fluidicallyisolated from other droplets, enabling accurate control of respectiveenvironments in the droplets.

Biological samples in partitions may be subjected to various processes,such as chemical processes or physical processes. Samples in partitionsmay be subjected to heating or cooling, or chemical reactions, such asto yield species that may be qualitatively or quantitatively processed.

SUMMARY

To gain further insight into the biology and pathology of cells, it maybe useful to capture and analyze multiple different types ofbiomolecules, such as ribonucleic acid (RNA) molecules anddeoxyribonucleic acid (DNA) molecules, from a single cell. However,different types of biomolecules may require, or be compatible with,different types of chemistry or processing mechanisms, and obtaining onetype of biomolecule from a cell may undesirably alter or destroy anothertype of biomolecule, or otherwise render it unobtainable. Therefore,recognized herein is a need to address at least the abovementionedproblems.

In an aspect, provided is a method for barcoding nucleic acid moleculesof a cell, comprising: (a) co-partitioning a cell bead and a first beadin a first partition, wherein the cell bead is generated from the cell,wherein the cell comprises a first type of nucleic acid molecule and asecond type of nucleic acid molecule, wherein the first type and thesecond type are different, and wherein the first bead comprises a firstset of barcode molecules each comprising a first nucleic acid barcodesequence; (b) subjecting the first partition to first conditionssufficient to permit (i) a first target nucleic acid molecule of thefirst type to couple to a first barcode molecule of the first set ofbarcode molecules to generate a first barcoded nucleic acid moleculecomprising the first nucleic acid barcode sequence, and (ii) a secondbarcode molecule of the first set of barcode molecules to associate withthe cell bead; (c) co-partitioning the cell bead associated with thesecond barcode molecule or derivative thereof and a second bead in asecond partition, wherein the second bead comprises a second set ofbarcode molecules, each comprising a second nucleic acid barcodesequence; and (d) subjecting the second partition to second conditionssufficient to permit (i) a second target nucleic acid molecule of thesecond type to couple to a third barcode molecule of the second set ofbarcode molecules to generate a second barcoded nucleic acid moleculecomprising the second nucleic acid barcode sequence, and (ii) a fourthbarcode molecule of the second set of barcode molecules to couple to thesecond barcode molecule to generate a composite barcode moleculecomprising the first nucleic acid barcode sequence and the secondnucleic acid barcode sequence.

In some embodiments, in (b), the second barcode molecule attaches to thecell bead.

In some embodiments, the method further comprises, subsequent to (b),releasing or recovering the first barcoded nucleic acid molecule orderivative thereof and the cell bead associated with the second barcodemolecule or derivative thereof from the first partition.

In some embodiments, the method further comprises, subsequent to (d),releasing or recovering the second barcoded nucleic acid molecule orderivative thereof and the composite barcode molecule from the secondpartition.

In some embodiments, the method further comprises, sequencing the firstbarcoded nucleic acid molecule or derivative thereof, the secondbarcoded nucleic acid molecule or derivative thereof, and the compositebarcode molecule or derivative thereof to generate sequencing reads, andusing the sequencing reads to associate the first target nucleic acidmolecule and the second target nucleic acid molecule as havingoriginated from the cell or cell bead.

In some embodiments, prior to (a), the cell is alive. In someembodiments, the first conditions comprise lysing the cell.

In some embodiments, prior to (a), the cell is lysed.

In some embodiments, prior to (a), target nucleic acid molecules of thefirst type are coupled to the cell bead. In some embodiments, the targetnucleic acid molecules of the first type are coupled to the cell beadvia one or more capture nucleic acid molecules configured to capture thetarget nucleic acid molecules of the first type. In some embodiments,the first type of nucleic acid molecule comprises messenger ribonucleicacid (mRNA) and the one or more capture nucleic acid molecules comprisea dT sequence.

In some embodiments, the cell bead comprises a first layer of polymernetwork and a second layer of polymer network. In some embodiments, thefirst layer of polymer network and the second layer of polymer networkare not covalently bound. In some embodiments, the first layer ofpolymer network and the second layer of polymer network areinterpenetrating. In some embodiments, the first layer of polymernetwork is configured to dissociate by a first dissolution mechanism andthe second layer of polymer network is configured to dissociate by asecond dissolution mechanism, wherein the first dissolution mechanismand the second dissolution mechanism are different.

In some embodiments, the first dissolution mechanism comprises areduction or oxidation reaction. In some embodiments, the seconddissolution mechanism comprises a reduction or oxidation reaction.

In some embodiments, the first dissolution mechanism is configured torelease target nucleic acid molecules of the first type from the cellbead. In some embodiments, the first dissolution mechanism is configuredto release the target nucleic acid molecules of the first type from thecell bead but not target nucleic acid molecules of the second type. Insome embodiments, the second dissolution mechanism is configured torelease target nucleic acid molecules of the second type from the cellbead.

In some embodiments, the first conditions comprise applying the firstdissolution mechanism. In some embodiments, the second conditionscomprise applying the second dissolution mechanism.

In some embodiments, the first layer of polymer network comprisesdisulfide crosslinking bonds.

In some embodiments, the second layer of polymer network comprisesdihydroxybutane.

In some embodiments, (c) comprises using a magnetic field to capture thecell bead associated with the second barcode molecule.

In some embodiments, the first type of nucleic acid molecule is selectedfrom the group consisting of deoxyribonucleic acid and ribonucleic acid.In some embodiments, the second type of nucleic acid molecule isselected from the group consisting of deoxyribonucleic acid andribonucleic acid.

In some embodiments, the first conditions and the second conditions aredifferent. In some embodiments, the first conditions comprise applying afirst dissolution mechanism and the second conditions comprise applyinga second dissolution mechanism, wherein the first dissolution mechanismand the second dissolution mechanism are different.

In some embodiments, the first dissolution mechanism comprises areduction or oxidation reaction. In some embodiments, the seconddissolution mechanism comprises a reduction or oxidation reaction. Insome embodiments, the first dissolution mechanism is configured torelease target nucleic acid molecules of the first type from the cellbead. In some embodiments, the first dissolution mechanism is configuredto release the target nucleic acid molecules of the first type from thecell bead but not target nucleic acid molecules of the second type.

In some embodiments, the first partition is a droplet. In someembodiments, the second partition is a droplet.

In some embodiments, the first bead is a gel bead. In some embodiments,the second bead is a gel bead.

Another aspect of the present disclosure provides a non-transitorycomputer readable medium comprising machine executable code that, uponexecution by one or more computer processors, implements any of themethods above or elsewhere herein.

Another aspect of the present disclosure provides a system comprisingone or more computer processors and computer memory coupled thereto. Thecomputer memory comprises machine executable code that, upon executionby the one or more computer processors, implements any of the methodsabove or elsewhere herein.

Additional aspects and advantages of the present disclosure will becomereadily apparent to those skilled in this art from the followingdetailed description, wherein only illustrative embodiments of thepresent disclosure are shown and described. As will be realized, thepresent disclosure is capable of other and different embodiments, andits several details are capable of modifications in various obviousrespects, all without departing from the disclosure. Accordingly, thedrawings and description are to be regarded as illustrative in nature,and not as restrictive.

INCORPORATION BY REFERENCE

All publications, patents, and patent applications mentioned in thisspecification are herein incorporated by reference to the same extent asif each individual publication, patent, or patent application wasspecifically and individually indicated to be incorporated by reference.To the extent publications and patents or patent applicationsincorporated by reference contradict the disclosure contained in thespecification, the specification is intended to supersede and/or takeprecedence over any such contradictory material.

BRIEF DESCRIPTION OF THE DRAWINGS

The novel features of the invention are set forth with particularity inthe appended claims. A better understanding of the features andadvantages of the present invention will be obtained by reference to thefollowing detailed description that sets forth illustrative embodiments,in which the principles of the invention are utilized, and theaccompanying drawings (also “Figure” and “FIG.” herein), of which:

FIG. 1 shows an example of a microfluidic channel structure forpartitioning individual biological particles.

FIG. 2 shows an example of a microfluidic channel structure fordelivering barcode carrying beads to droplets.

FIG. 3 shows an example of a microfluidic channel structure forco-partitioning biological particles and reagents.

FIG. 4 shows an example of a microfluidic channel structure for thecontrolled partitioning of beads into discrete droplets.

FIG. 5 shows an example of a microfluidic channel structure forincreased droplet generation throughput.

FIG. 6 shows another example of a microfluidic channel structure forincreased droplet generation throughput.

FIG. 7A shows a cross-section view of another example of a microfluidicchannel structure with a geometric feature for controlled partitioning.FIG. 7B shows a perspective view of the channel structure of FIG. 7A.

FIG. 8 illustrates an example of a barcode-carrying bead.

FIG. 9 illustrates an example of a method for barcoding nucleic acidmolecules in a cell encapsulated in a cell bead.

FIG. 10 illustrates an example of a method for barcoding nucleic acidmolecules in a cell that is encapsulated and lysed in a cell bead.

FIG. 11 illustrates an example of a method for barcoding nucleic acidmolecules in a cell that is encapsulated and lysed in a cell beadcomprising an interpenetrating network.

FIG. 12 shows a computer system that is programmed or otherwiseconfigured to implement methods provided herein.

DETAILED DESCRIPTION

While various embodiments of the invention have been shown and describedherein, it will be obvious to those skilled in the art that suchembodiments are provided by way of example only. Numerous variations,changes, and substitutions may occur to those skilled in the art withoutdeparting from the invention. It should be understood that variousalternatives to the embodiments of the invention described herein may beemployed.

Where values are described as ranges, it will be understood that suchdisclosure includes the disclosure of all possible sub-ranges withinsuch ranges, as well as specific numerical values that fall within suchranges irrespective of whether a specific numerical value or specificsub-range is expressly stated.

The term “barcode,” as used herein, generally refers to a label, oridentifier, that conveys or is capable of conveying information about ananalyte. A barcode can be part of an analyte. A barcode can beindependent of an analyte. A barcode can be a tag attached to an analyte(e.g., nucleic acid molecule) or a combination of the tag in addition toan endogenous characteristic of the analyte (e.g., size of the analyteor end sequence(s)). A barcode may be unique. Barcodes can have avariety of different formats. For example, barcodes can include:polynucleotide barcodes; random nucleic acid and/or amino acidsequences; and synthetic nucleic acid and/or amino acid sequences. Abarcode can be attached to an analyte in a reversible or irreversiblemanner. A barcode can be added to, for example, a fragment of adeoxyribonucleic acid (DNA) or ribonucleic acid (RNA) sample before,during, and/or after sequencing of the sample. Barcodes can allow foridentification and/or quantification of individual sequencing-reads.

The term “real time,” as used herein, can refer to a response time ofless than about 1 second, a tenth of a second, a hundredth of a second,a millisecond, or less. The response time may be greater than 1 second.In some instances, real time can refer to simultaneous or substantiallysimultaneous processing, detection or identification.

The term “subject,” as used herein, generally refers to an animal, suchas a mammal (e.g., human) or avian (e.g., bird), or other organism, suchas a plant. For example, the subject can be a vertebrate, a mammal, arodent (e.g., a mouse), a primate, a simian or a human. Animals mayinclude, but are not limited to, farm animals, sport animals, and pets.A subject can be a healthy or asymptomatic individual, an individualthat has or is suspected of having a disease (e.g., cancer) or apre-disposition to the disease, and/or an individual that is in need oftherapy or suspected of needing therapy. A subject can be a patient. Asubject can be a microorganism or microbe (e.g., bacteria, fungi,archaea, viruses).

The term “genome,” as used herein, generally refers to genomicinformation from a subject, which may be, for example, at least aportion or an entirety of a subject’s hereditary information. A genomecan be encoded either in DNA or in RNA. A genome can comprise codingregions (e.g., that code for proteins) as well as non-coding regions. Agenome can include the sequence of all chromosomes together in anorganism. For example, the human genome ordinarily has a total of 46chromosomes. The sequence of all of these together may constitute ahuman genome.

The terms “adaptor(s)”, “adapter(s)” and “tag(s)” may be usedsynonymously. An adaptor or tag can be coupled to a polynucleotidesequence to be “tagged” by any approach, including ligation,hybridization, or other approaches.

The term “sequencing,” as used herein, generally refers to methods andtechnologies for determining the sequence of nucleotide bases in one ormore polynucleotides. The polynucleotides can be, for example, nucleicacid molecules such as deoxyribonucleic acid (DNA) or ribonucleic acid(RNA), including variants or derivatives thereof (e.g., single-strandedDNA). Sequencing can be performed by various systems currentlyavailable, such as, without limitation, a sequencing system byIllumina®, Pacific Biosciences (PacBio®), Oxford Nanopore®, or LifeTechnologies (Ion Torrent®). Alternatively or in addition, sequencingmay be performed using nucleic acid amplification, polymerase chainreaction (PCR) (e.g., digital PCR, quantitative PCR, or real time PCR),or isothermal amplification. Such systems may provide a plurality of rawgenetic data corresponding to the genetic information of a subject(e.g., human), as generated by the systems from a sample provided by thesubject. In some examples, such systems provide sequencing reads (also“reads” herein). A read may include a string of nucleic acid basescorresponding to a sequence of a nucleic acid molecule that has beensequenced. In some situations, systems and methods provided herein maybe used with proteomic information.

The term “bead,” as used herein, generally refers to a particle. Thebead may be a solid or semi-solid particle. The bead may be a gel bead.The gel bead may include a polymer matrix (e.g., matrix formed bypolymerization or cross-linking). The polymer matrix may include one ormore polymers (e.g., polymers having different functional groups orrepeat units). Polymers in the polymer matrix may be randomly arranged,such as in random copolymers, and/or have ordered structures, such as inblock copolymers. Cross-linking can be via covalent, ionic, orinductiveinteractions, or physical entanglement. The bead may comprise amacromolecule. The bead may comprise a sol-gel. The bead may be formedof nucleic acid molecules bound together. The bead may be formed viacovalent or non-covalent assembly of molecules (e.g., macromolecules),such as monomers or polymers. Such polymers or monomers may be naturalor synthetic. Such polymers or monomers may be or include, for example,nucleic acid molecules (e.g., DNA or RNA). The bead may be formed of apolymeric material. The bead may be magnetic or non-magnetic. The beadmay be rigid. The bead may be flexible and/or compressible. The bead maybe disruptable or dissolvable. The bead may be a solid particle (e.g., ametal-based particle including but not limited to iron oxide, gold orsilver) covered with a coating comprising one or more polymers. Suchcoating may be disruptable or dissolvable.

The term “sample,” as used herein, generally refers to a biologicalsample of a subject. The biological sample may comprise any number ofmacromolecules, for example, cellular macromolecules. The sample may bea cell sample. The sample may be a cell line or cell culture sample. Thesample can include one or more cells. The sample can include one or moremicrobes. The biological sample may be a nucleic acid sample or proteinsample. The biological sample may also be a carbohydrate sample or alipid sample. The biological sample may be derived from another sample.The sample may be a tissue sample, such as a biopsy, core biopsy, needleaspirate, or fine needle aspirate. The sample may be a fluid sample,such as a blood sample, urine sample, or saliva sample. The sample maybe a skin sample. The sample may be a cheek swab. The sample may be aplasma or serum sample. The sample may be a cell-free sample. Acell-free sample may include extracellular polynucleotides.Extracellular polynucleotides may be isolated from a bodily sample thatmay be selected from the group consisting of blood, plasma, serum,urine, saliva, mucosal excretions, sputum, stool and tears.

The term “biological particle,” as used herein, generally refers to adiscrete biological system derived from a biological sample. Thebiological particle may be a macromolecule. The biological particle maybe a small molecule. The biological particle may be a virus. Thebiological particle may be a cell or derivative of a cell. Thebiological particle may be an organelle. The biological particle may bea rare cell from a population of cells. The biological particle may beany type of cell, including without limitation prokaryotic cells,eukaryotic cells, bacterial, fungal, plant, mammalian, or other animalcell type, mycoplasmas, normal tissue cells, tumor cells, or any othercell type, whether derived from single cell or multicellular organisms.The biological particle may be a constituent of a cell. The biologicalparticle may be or may include DNA, RNA, organelles, proteins, or anycombination thereof. The biological particle may be or may include amatrix (e.g., a gel or polymer matrix) comprising a cell or one or moreconstituents from a cell (e.g., cell bead), such as DNA, RNA,organelles, proteins, or any combination thereof, from the cell. Thebiological particle may be obtained from a tissue of a subject. Thebiological particle may be a hardened cell. Such hardened cell may ormay not include a cell wall or cell membrane. The biological particlemay include one or more constituents of a cell, but may not includeother constituents of the cell. An example of such constituents is anucleus or an organelle. A cell may be a live cell. The live cell may becapable of being cultured, for example, being cultured when enclosed ina gel or polymer matrix, or cultured when comprising a gel or polymermatrix.

The term “macromolecular constituent,” as used herein, generally refersto a macromolecule contained within or from a biological particle. Themacromolecular constituent may comprise a nucleic acid. In some cases,the biological particle may be a macromolecule. The macromolecularconstituent may comprise DNA. The macromolecular constituent maycomprise RNA. The RNA may be coding or non-coding. The RNA may bemessenger RNA (mRNA), ribosomal RNA (rRNA) or transfer RNA (tRNA), forexample. The RNA may be a transcript. The RNA may be small RNA that areless than 200 nucleic acid bases in length, or large RNA that aregreater than 200 nucleic acid bases in length. Small RNAs may include5.8S ribosomal RNA (rRNA), 5S rRNA, transfer RNA (tRNA), microRNA(miRNA), small interfering RNA (siRNA), short hairpin RNA (shRNA), smallnucleolar RNA (snoRNAs), Piwi-interacting RNA (piRNA), tRNA-derivedsmall RNA (tsRNA) and small rDNA-derived RNA (srRNA). The RNA may bedouble-stranded RNA or single-stranded RNA. The RNA may be circular RNA.The macromolecular constituent may comprise a protein. Themacromolecular constituent may comprise a peptide. The macromolecularconstituent may comprise a polypeptide.

The term “molecular tag,” as used herein, generally refers to a moleculecapable of binding to a macromolecular constituent. The molecular tagmay bind to the macromolecular constituent with high affinity. Themolecular tag may bind to the macromolecular constituent with highspecificity or selectivity. The molecular tag may comprise a nucleotidesequence. The molecular tag may comprise a nucleic acid sequence. Thenucleic acid sequence may be at least a portion or an entirety of themolecular tag. The molecular tag may be a nucleic acid molecule or maybe part of a nucleic acid molecule. The molecular tag may be anoligonucleotide or a polypeptide. The molecular tag may comprise a DNAaptamer. The molecular tag may be or comprise a primer. The moleculartag may be, or comprise, a protein. The molecular tag may comprise apolypeptide. The molecular tag may be a barcode.

The term “partition,” as used herein, generally, refers to a space orvolume that may be suitable to contain one or more species or conductone or more reactions. A partition may be a physical compartment, suchas a droplet or well. The partition may isolate space or volume fromanother space or volume. The droplet may be a first phase (e.g., aqueousphase) in a second phase (e.g., oil) immiscible with the first phase.The droplet may be a first phase in a second phase that does not phaseseparate from the first phase, such as, for example, a capsule orliposome in an aqueous phase. A partition may comprise one or more other(inner) partitions. In some cases, a partition may be a virtualcompartment that can be defined and identified by an index (e.g.,indexed libraries) across multiple and/or remote physical compartments.For example, a physical compartment may comprise a plurality of virtualcompartments.

The terms “dissolution,” “disassociation” and “dissociation” may be usedinterchangeably herein and generally refer to the separation,disconnection, and/or splitting of one or more species from the same ordifferent one or more species.

The terms “a,” “an,” and “the,” as used herein, generally refers tosingular and plural references unless the context clearly dictatesotherwise.

Methods for DNA and RNA Barcoding

Provided herein are methods for barcoding nucleic acid molecules of acell. The method can comprise co-partitioning a cell bead and a firstbead in a first partition, wherein the cell bead is generated from thecell, wherein the cell comprises a first type of nucleic acid moleculeand a second type of nucleic acid molecule, wherein the first type andthe second type are different, and wherein the first bead comprises afirst set of barcode molecules, each comprising a first nucleic acidbarcode sequence. The first partition can then be subject to firstconditions sufficient to permit (i) a first target nucleic acid moleculeof the first type to couple to a first barcode molecule of the first setof barcode molecules to generate a first barcoded nucleic acid moleculecomprising the first nucleic acid barcode sequence, and (ii) a secondbarcode molecule of the first set of barcode molecules to associate withthe cell bead. Then, the cell bead associated with the second barcodemolecule or derivative thereof can be co-partitioned with a second beadin a second partition, wherein the second bead comprises a second set ofbarcode molecules, each comprising a second nucleic acid barcodesequence. The second partition can then be subject to second conditionssufficient to permit (i) a second target nucleic acid molecule of thesecond type to couple to a third barcode molecule of the second set ofbarcode molecules to generate a second barcoded nucleic acid moleculecomprising the second nucleic acid barcode sequence, and (ii) a fourthbarcode molecule of the second set of barcode molecules to couple to thesecond barcode molecule to generate a composite barcode moleculecomprising the first nucleic acid barcode sequence and the secondnucleic acid barcode sequence.

In some embodiments, a cell selected for generation of a cell bead is aeukaryotic cell (e.g., fungal, yeast, insect, and mammalian cells).Non-limiting examples of mammalian cells include HEK293 cells, HeLacells, CHO cells, COS cells, Jurkat cells, U20 cells, U251 cells, MCF7cells, SKBR3 cells, NS0 hybridoma cells, baby hamster kidney (BHK)cells, MDCK cells, NIH-3T3 fibroblast cells, and any other immortalizedcell line derived from a mammalian cell or mammalian cells derived fromprimary cell culture. In some embodiments, a cell selected forgeneration of a cell bead is a prokaryotic cell (e.g., archaeal cells,bacterial cells). A cell may be alive or dead (e.g., fixed,permeabilized, or lysed) when the cell bead is generated.

The nucleic acid molecule may be, for example, deoxyribonucleic acid(DNA) or ribonucleic acid (RNA), including variants or derivativesthereof, described elsewhere herein. For example, the first type ofnucleic acid molecule may be DNA and the second type of nucleic acidmolecule may be RNA, or vice versa. In another example, the first typeof nucleic acid molecule may comprise messenger RNA (mRNA) and thesecond type of nucleic acid molecule may comprise non-mRNA molecules.

A cell bead can be any cell bead, such as a bead that containsbiological particles (e.g., a cell) or macromolecular constituents(e.g., RNA, DNA, proteins, etc.) of biological particles. See, e.g.,U.S. Pat. Pub. 2018/0216162 and U.S. Pat. Pub. 2019/0100632, which areincorporated by reference in their entirety, for exemplary cell beadcompositions, generation systems, and methods. A cell bead may include asingle cell or multiple cells, or a derivative of the single cell ormultiple cells. For example, after lysing and washing the cells,inhibitory components from cell lysates can be washed away and themacromolecular constituents can be bound as cell beads. Systems andmethods disclosed herein can be applicable to both cell beads (and/ordroplets or other partitions) containing biological particles and cellbeads (and/or droplets or other partitions) containing macromolecularconstituents of biological particles.

In some cases, a cell bead may comprise capture nucleic acid moleculesconfigured to couple to a barcode nucleic acid molecule. See, e.g., U.S.Pat. Pub. 2019/0100632 and U.S. Pat. Application 16/434,095 filed Jun.6, 2019, which are both incorporated by reference in their entirety, forexemplary cell bead polymers, functionalization strategies, and nucleicacid molecules for generating cell beads comprising, e.g., nucleic acidmolecules. In some cases, the capture nucleic acid molecules maycomprise barcode nucleic acid molecules (e.g., barcode RNA molecule,barcode DNA molecule, or barcode RNA-DNA molecule) that comprise asequence that can hybridize with other barcode or non-barcode nucleicacid molecules. The capture nucleic acid molecules or barcode nucleicacid molecules may be bound to the cell bead through, in non-limitingexamples, covalent or ionic bonds, or through hydrophobic (e.g., van derWaals) interactions.

A bead may be porous, non-porous, solid, semi-solid, semi-fluidic,fluidic, and/or a combination thereof. In some instances, a bead may bedissolvable, disruptable, and/or degradable. In some cases, a bead maynot be degradable. In some cases, the bead may be a gel bead. A gel beadmay be a hydrogel bead and comprise derivatives or variants thereof,described elsewhere herein.

In some cases, the beads (e.g., cell bead, gel bead) may comprise one ormore crosslinking types that are dissolvable or dissociable. In someinstances, as described elsewhere herein, nucleic acid barcode moleculesattached to a bead may comprise one or more labile bonds releasable uponapplication of a stimulus. For example, a bead may comprise disulfidebonds, where upon exposure to a reducing agent, the bead may dissolveand/or dissociate. In another example, a bead may comprisedihydroxybutane crosslinks. Dissolution of the bead may occur byexposure to oxidation reagents, e.g., sodium periodate.

In some cases, a cell bead may be co-partitioned with a first bead(e.g., a gel bead as described elsewhere herein) comprising a first setof barcode nucleic acid molecules. Upon or following partitioning, thefirst set of barcode nucleic acid molecules from the first bead may bereleased, e.g., via a first dissolution or releasing mechanism.Dissolution mechanisms may comprise reducing agents (e.g., DTT, TCEP,etc.) and/or oxidizing agents (e.g., sodium periodate) or derivatives orvariations thereof, as described elsewhere herein. The first set ofbarcode nucleic acid molecules may each comprise a first barcodesequence, for example, common to the first bead.

A subset of the first set of barcode nucleic acid molecules may enterthe cell bead and associate (e.g., hybridize) with the bound capturenucleic acid molecules. The subset of the first set of barcode nucleicacid molecule may henceforth be bound to the cell bead. For example, insome instances, the capture nucleic acid molecules bound to the cellbead (which may be releasable or non-releasable, see, e.g., U.S. Pat.Pub. 2019/0100632, which is incorporated by reference in its entirety)may comprise a sequence complementary to a sequence on the nucleic acidbarcode molecules on the first bead. Furthermore, the capture nucleicacid molecules may comprise one or more functional sequences such as abarcode sequence, a unique molecular identifier (UMI), a primer sequenceor primer binding sequence (e.g., a sequencing primer sequence (orpartial sequencing primer sequence) such as an R1 and/or R2 sequence), asequence configured to attach to the flow cell of a sequencer (e.g., P5and/or P7). In some cases, upon or following partitioning, the cell maybe lysed to release intracellular contents (e.g., proteins, mRNA) intothe cell bead. In some cases, the intracellular contents may exit thecell bead and enter another region of the first partition (e.g.,droplet). See, e.g., See, e.g., U.S. Pat. 10,011,872, which isincorporated by reference in its entirety, for exemplary molecules andmethods for analyzing protein molecules and other analytes using, e.g.,barcoded labelling agents. In some cases, the cell nucleus may remainintact. In some cases, cell lysis may not maintain the nucleus intact.In some cases, a first type of nucleic acid molecule (e.g., DNA) fromthe cell may be retained in the cell bead while a portion or all of asecond type of nucleic acid molecule (e.g., RNA) exits the cell beadand/or is released into the partition (e.g., droplet). In some cases,one or more types of nucleic acid molecules may be retained in the cellbead. In some cases, a portion of one or more types of nucleic acidmolecules may be retained in the cell bead.

Cellular nucleic acid molecules released into the first partition may bebarcoded, e.g., via hybridization with another subset of the first setof barcode nucleic acid molecules released from the first gel bead, togenerate a first set of barcoded nucleic acid molecules. The barcodednucleic acid molecules may then be collected from the partition whilemaintaining the cell bead intact.

The cell bead may be retrieved from the first partition and prepared fora second partitioning. Retrieval of the cell bead may occur through asorting mechanism. In one non-limiting example, the cell bead can bemagnetic and/or comprise magnetic particles. The cell bead may beretrieved by application of a magnetic field. Other non-limitingexamples of retrieval mechanisms comprise biochemical mechanisms (e.g.,incorporation of antigens and retrieval via antibodies) and/or physicalmechanisms (e.g., optical tweezers).

In some cases, in a second partition, the cell bead may beco-partitioned with a second gel bead comprising a second set of barcodenucleic acid molecules. The second set of barcode nucleic acid moleculesmay be a different type of nucleic acid molecule than the first set ofbarcode nucleic acid molecules. The second set of barcode nucleic acidmolecules may each comprise a second barcode sequence, for example,common to the second gel bead. The second barcode sequence may bedifferent from the first barcode sequence. Upon or followingpartitioning, the second set of barcode nucleic acid molecules from thesecond gel bead may be released via a second dissolution mechanism.Simultaneously or thereafter, the cell bead may also be dissolved.Biomolecules retained in the cell bead may be released (e.g.,intracellular and/or intra-nuclear proteins, DNA) into the secondpartition. The first set of barcode nucleic acid molecules bound to thecell bead may also be released. The released nucleic acid molecules(e.g., DNA) and the first set of barcode nucleic acid molecules, may bebarcoded, e.g., via hybridization, with the second set of barcodenucleic acid molecules. The first set of barcode nucleic acid moleculesbound to the cell bead may be barcoded with the second set of barcodednucleic acid molecules to generate composite barcode molecules. Thecomposite barcode molecules may each comprises a composite barcodesequence including both the first barcode sequence and the secondbarcode sequence. The released contents of the cell in the secondpartition (e.g., DNA, proteins) may be barcoded with the second set ofbarcoded nucleic acid molecules to generate a second set of barcodednucleic acid molecules. The nucleic acid molecules released from thecell bead in the first partition and the second partition, respectively,may be of different types (e.g., RNA, DNA).

The components of the second partition may be collected. The second setof barcoded nucleic acid molecules or derivative thereof and thecomposite barcode molecules may be collected from the second partition.

Subsequent to collection, the barcoded nucleic acid molecules and/orderivatives thereof may be sequenced. For example, the first set ofbarcoded molecules from the first partition, the second set of barcodedmolecule from the second partition, and the composite barcode moleculesfrom the second partition may be sequenced. The sequencing readsgenerated may be used to associate the nucleic acid molecules with theorigin of the nucleic acid molecules (e.g., from the cell or cell bead).For example, the first barcode sequence may be used to associate thefirst type of nucleic acid molecules to a cell, cell bead, or partition;the second barcode sequence may be used to associate the second type ofnucleic acid nucleic acid molecules to the same or different cell, cellbead, or partition, and the composite barcode sequence may be used toassociate the first type of nucleic acid molecule and the second type ofnucleic acid molecule as arising from the same cell or cell bead.

In some embodiments, cell lysis may occur in the cell bead prior toco-partitioning. The intracellular contents (e.g., RNA, proteins) may becaptured within and/or coupled to the surface of the cell bead bycapture molecules (e.g., capture nucleic acid molecules) bound to thepolymer matrix of the cell bead. In one non-limiting example, cellularmRNA may be captured by poly-T nucleic acids that are attached to thepolymer matrix. Attachment of the capture molecules to the polymermatrix may occur through a variety of cross-links or binding mechanisms,e.g., acrydite-conjugated nucleic acids, NHS-ester linkages ofantibodies, and may include variants or derivatives thereof, describedelsewhere herein.

FIG. 9 illustrates an example of a method 900 for barcoding nucleic acidmolecules. As shown in panel A, a cell bead 902 may comprise a singlecell 904. Capture nucleic acid molecules (not shown), which mayoptionally comprise a barcode sequence, may be bound to the cell bead902. Panel B shows a first partition (e.g., droplet) 906 comprising thecell bead 902 and a gel bead 910 comprising a first set of barcodenucleic acid molecules 914, each of which comprise a first barcodesequence. The first set of barcode nucleic acid molecules may eachcomprise a first barcode sequence. The first partition can comprise, orbe subjected to, conditions sufficient to permit the first set ofbarcode nucleic acid molecules (e.g., barcode RNA molecules) 914 to bereleased from the gel bead 910. The first partition may also comprise,or be subject to, conditions sufficient to permit release ofintracellular contents from the cell bead 902, such as a first type ofnucleic acid molecule (e.g., RNA). For example, such conditions maycomprise lysis of the cell 904. The conditions sufficient to release thefirst type of nucleic acid molecules from the cell bead 902 may retainthe integrity of the cell bead 902. In some instances, the conditionssufficient to release the first type of nucleic acid molecules from thecell bead 902 may be the same conditions sufficient to release the firstset of barcode nucleic acid molecules 914 from the first gel bead 910.The first type of nucleic acid molecules (e.g., RNA) may also associatewith the first set of barcode nucleic acid molecules 914 from the gelbead 910 and form a first set of barcoded nucleic acid molecules 912(e.g., barcoded RNA). The released first set of barcode nucleic acidmolecules 914 may also enter the cell bead 902 and associate with thecapture nucleic acid molecules in the cell bead, forming a second set ofbarcoded nucleic acid molecules 916 that are associated with the cellbead 902. The cell bead 902 comprising the second set of barcodednucleic acid molecules 916 may be collected. Also, the first set ofbarcoded nucleic acid molecules (e.g., barcoded RNA molecules) 912 maybe collected and may be sequenced. Panel C shows a second partition(e.g., droplet) 918 comprising the cell bead 902 (e.g., collected fromthe first partition) and a second gel bead 922 comprising a second setof barcode nucleic acid molecules (e.g., barcode DNA molecules) 924,each comprising a second barcode sequence. The second set of barcodenucleic acid molecules may each comprise a second barcode sequence,which may be the same or different than the first barcode sequence. Thesecond partition may comprise, or be subject to, conditions sufficientto permit the second set of barcode nucleic acid molecules 924 to bereleased from the gel bead 922. The second partition may also comprise,or be subject to, conditions sufficient to release a second type ofnucleic acid molecule (e.g., DNA) 920 from the cell bead 902. In someinstances, the conditions sufficient to release the second type ofnucleic acid molecule 920 from the cell bead 902 may comprisedisassociation of the cell bead 902. In some instances, the conditionssufficient to release the second type of nucleic acid molecule 920 fromthe cell bead 902 may be the same conditions sufficient to release thesecond set of barcode nucleic acid molecules 924 from the second gelbead 922. The released second set of barcode nucleic acid molecules 924may associate with the released second type of nucleic acid molecule(e.g., DNA) 920 from the cell bead 902, forming a third set of barcodednucleic acid molecule (e.g., barcoded DNA not shown). The releasedsecond set of barcode nucleic acid molecules 924 may also associate withthe released, second set of barcoded nucleic acid molecule 916, forminga set of composite barcoded molecules (not shown). The method 900 mayalso comprise collecting the first set of barcoded nucleic acidmolecules (e.g., barcoded RNA), comprising the first barcode sequence,the third set of barcoded nucleic acid molecules (e.g., barcoded DNA),comprising the second barcode sequence, and the set of compositebarcoded molecules, comprising both the first barcode sequence and thesecond barcode sequence. Upon sequencing, the first barcode sequence maybe identified and used to associate the first type of nucleic acidmolecules (e.g., RNA) to a cell, cell bead, or partition; the secondbarcode sequence may be identified and used to associate the second typeof nucleic acid nucleic acid molecules (e.g., DNA) to the same ordifferent cell, cell bead, or partition, and the composite barcodesequence may be identified and used to associate the first type ofnucleic acid molecule and the second type of nucleic acid molecule asarising from the same cell or cell bead.

FIG. 10 illustrates another example of a method 1000 for barcodingnucleic acid molecules. As shown in Panel A, a cell bead 1002 maycomprise a single cell 1004. Capture nucleic acid molecules (whichoptionally may comprise a barcode sequence, not shown) may be bound tothe cell bead 1002. In some cases, the cell is lysed prior toco-partitioning, but the nucleus may remain intact. The intracellularcontents, such as nucleic acids including the first type of nucleic acidmolecule (e.g., RNA), may associate with the cell bead matrix (e.g., viathe capture nucleic acid molecules, which may comprise poly-T sequencesthat are incorporated in the cell bead matrix, e.g., viaacrydite-modification), forming associated nucleic acid molecules 1006.Panel B shows a first partition (droplet) 1008 comprising the cell bead1002 and a gel bead 1010 comprising a first set of barcode nucleic acidmolecules (e.g., barcode RNA molecules) 1016, each comprising a firstbarcode sequence. The first partition comprises conditions sufficient topermit the first set of barcode nucleic acid molecules (e.g., barcodeRNA molecules) 1016 to be released from the gel bead 1012. The firstpartition may also comprise conditions sufficient to permit lysis of thecell 1004 and release of intracellular contents from the cell bead 1010,such as the first type of nucleic acid molecule (e.g., RNA, mRNAhybridized with the capture poly-T nucleic acid sequence, etc.). Thefirst type of nucleic acid molecules (e.g., RNA) may also associate withthe barcode nucleic acid molecules from the gel bead 1012 and form afirst set of barcoded nucleic acid molecules 1014 (e.g., barcoded RNAmolecules). The released first set of barcode nucleic acid molecules1016 may also enter the cell bead 1002 and associate with a second typeof capture molecule, which may be configured to couple to nucleic acidmolecules, thereby forming a second set of barcoded nucleic acidmolecules 1018. The cell bead 1010 comprising the second set of barcodednucleic acid molecules associated with the cell bead 1014 may becollected. Also, the first set of barcoded nucleic acid molecules (e.g.,barcoded RNA molecules) 1014 may be collected and may be sequenced.Panel C shows a second partition (droplet) 1020 comprising a cell bead(e.g., 1010 collected from the first partition) and a second gel bead1024 comprising a second set of barcode nucleic acid molecules (e.g.,barcode DNA molecules) 1026, each of which comprise a second barcodesequence. The second partition comprises conditions sufficient to permitthe second set of barcode nucleic acid molecules 1026 to be releasedfrom the gel bead 1024. The second partition may also compriseconditions sufficient to release a second type of nucleic acid molecule(e.g., DNA) 1022 from the cell bead. The released second set of barcodenucleic acid molecules 1026 may associate with the released second typeof nucleic acid molecule (e.g., DNA) 1022 from the cell bead, forming athird set of barcoded nucleic acid molecules. The released second set ofbarcode nucleic acid molecules 1026 may also associate with thereleased, second set of barcoded nucleic acid molecules 1018, forming acomposite barcoded molecule (e.g., barcoded RNA-DNA) comprising thefirst barcode sequence and the second barcode sequence.

FIG. 11 illustrates another example of a method 1100 for barcodingnucleic acid molecules. As shown in panel A, a cell bead 1102 maycomprise a single cell 1104. Capture nucleic acid molecules (not shown)may be bound to the cell bead 1104. In some cases, the cell bead 1102comprises an interpenetrating network (IPN) of two or more polymermeshes. Each polymer mesh may be dissociable via different mechanisms.In one example, one polymer network of the IPN may comprise disulfidecrosslinks or bonds that may be dissociated when reducing agents (e.g.,DTT) are introduced. A second polymer network of the IPN may comprisedihydroxybutane crosslinks or bonds that are dissociable when oxidizingagents (e.g., sodium periodate) are introduced. The pore size of the IPNmay be sufficiently small to prevent passage of intracellular contents(e.g., proteins, RNA). In some cases, the cell 1104 is lysed in the cellbead. Panel B shows a first partition (droplet) 1108 comprising the cellbead 1102 and a gel bead 1112 comprising a first set of barcode nucleicacid molecules (e.g., barcode RNA) 1116, each of which comprises a firstbarcode sequence. The first partition (droplet) may comprise conditionssufficient to permit the first barcode nucleic acid molecules (e.g.,barcode RNA) 1116 to be released from the gel bead 1112 and also fordissociation of one or more of the polymer meshes in the IPN. Thedissociation of one or more of the polymer meshes in the IPN mayincrease the pore size of the cell bead 1102 such that some componentsof the intracellular contents (e.g., a first type of nucleic acidmolecule, e.g., RNA) can exit the cell bead into the first partition.

The first partition may also comprise conditions sufficient to permitlysis of the cell 1104 and release of intracellular contents from thecell bead 1110, such as the first type of nucleic acid molecule (e.g.,RNA). The first type of nucleic acid molecules (e.g., RNA) may associatewith the first set of barcode nucleic acid molecules 1116 from the gelbead 1112 and form a first set of barcoded nucleic acid molecules 1114.The released first set of barcode nucleic acid molecules 1116 may alsoenter the cell bead 1110 and associate with capture nucleic acidmolecules in the cell bead, forming a second set of barcoded nucleicacid molecules 1118. The cell bead 1110 comprising the second set ofbarcoded nucleic acid molecules 1118 may be collected. Also, the firstset of barcoded nucleic acid molecules (e.g., barcoded RNA molecules)1114 may be collected and may be sequenced. Panel C shows a secondpartition (droplet) 1120 comprising a cell bead (e.g., 1102 collectedfrom the first partition) and a second gel bead 1124 comprising a secondset of barcode nucleic acid molecules (e.g., barcode DNA molecules)1126, each of which comprises a second barcode sequence. The secondpartition comprises conditions sufficient to permit the second set ofbarcode nucleic acid molecules 1126 to be released from the gel bead1124. The second partition may also comprise conditions to dissociatethe second polymer network to release a second type of nucleic acidmolecule (e.g., DNA) 1122 from the cell bead. The released second set ofbarcode nucleic acid molecules 1126 may associate with the releasedsecond type of nucleic acid molecule (e.g., DNA) 1122 from the cellbead, forming a third set of barcoded nucleic acid molecule. Thereleased second set of barcode nucleic acid molecules 1126 may alsoassociate with the released, first set of barcoded nucleic acidmolecules 1118, forming a composite barcoded molecule comprising thefirst barcode sequence and the second barcode sequence.

The barcoded nucleic acid molecules may be subjected to furtherprocessing and analysis, e.g., sequencing. Sequencing may be used togenerate sequencing reads, which can be used to associate barcodesequences with a partition or cell bead. For example, the first barcodenucleic acid molecule may comprise a first barcode sequence. Asdescribed herein, the first barcode nucleic acid molecule may associatewith (i) the first type of nucleic acid molecule (e.g., RNA) to form afirst barcoded nucleic acid molecule comprising the first barcodesequence and (ii) the cell bead (e.g., via a capture sequence) to form asecond barcoded nucleic acid molecule, also comprising the first barcodesequence. The first barcoded nucleic acid molecule may be subjected tosequencing. The cell bead, comprising the second barcoded nucleic acidmolecule, may be partitioned with a second barcode nucleic acid moleculecomprising a second barcode sequence, which may be a different sequencethan the first barcode sequence. The second barcode nucleic acidmolecule may associate with (i) the second type of nucleic acid molecule(e.g., DNA) to form a third barcoded nucleic acid molecule and (ii) thesecond barcoded nucleic acid molecule from the cell bead to generate thecomposite barcode molecule, comprising the first barcode sequence andthe second barcode sequence. From sequencing all the barcoded products,the origin of the products may be determined. For example, the firstbarcode sequence may be used to determine that the first type of nucleicacid molecule (e.g., RNA) arose from a specified cell bead, which cellbead comprises the composite barcode comprising the first (and thesecond) barcode sequence. The second barcode sequence may be used todetermine that the second type of nucleic acid molecule (e.g., DNA)arose from the same specified cell bead, as the cell bead comprises thecomposite barcode comprising the second (and the first) barcodesequence. Thus, both types of nucleic acid molecules may be associatedwith a single cell.

In some embodiments, a cell bead can comprise one or more polymernetworks. These polymer networks may form one or more interpenetratingnetworks (IPNs). An IPN may comprise two or more polymer networks and/ormeshes which are at least partially interlaced but not covalently bondedto one another. In some embodiments, the IPNs arefully-interpenetrating, semi-interpenetrating, orpseudo-interpenetrating. An IPN may comprise one or more polymers,including, in non-limiting examples, polyacrylamide, alginate,poly-lactic acid (PLA), poly-ethylene glycol(PEG), PEG-diol,PEG-diacrylate (PEGDA), and other synthetic and/or natural polymers, asdescribed elsewhere herein. In some cases, one or both polymer networksmay be crosslinked using crosslinks that may be dissolved and/ordissociated. In non-limiting examples, one of the polymer networks maybe cross-linked with disulfide bonds while a second polymer network maybe crosslinked with dihydroxybutane bonds. In some cases, reduction(e.g., with DTT) may disrupt and/or dissolve the disulfide bonds, whilea separate oxidation step may be used to disrupt and/or dissolve thedihydroxybutane bonds. See, e.g., U.S. Pat. Pub. 2018/0216162 and U.S.Pat. Pub. 2019/0100632, which are incorporated by reference in theirentirety, for exemplary cell bead polymers, polymer functionalization,and strategies for generating cell beads.

The IPN may have various tunable properties, e.g., physical, mechanical,chemical, biological, and biochemical. Non-limiting examples of saidproperties include: pore size, stiffness, ligand density, charge,hydrophobicity, etc. In some cases, one or more properties of IPNs maybe tuned by association or dissociation of one or more of the polymernetworks.

In some cases, the IPN can comprise two layers of polymer networks thatmay be configured to dissociate by two different dissolution mechanisms.The dissolution mechanisms may be configured such that one dissolutionmechanism may dissolve and/or dissociate one of the two polymer networksbut not the other. For example, a second dissolution mechanism may beconfigured to dissolve the second polymer network, but not the first,and vice-versa. The separate dissolution mechanisms may be used toselectively release different types of nucleic acid molecules.

In some cases, the IPN may be used to selectively entrap biomolecules(e.g., proteins, lipids, nucleic acids, polysaccharides). In onenon-limiting example, the pore size of the IPN may be tuned to inhibitthe entrance and exit of biomolecules, such as RNA and DNA. In somecases, the dissociation of one or more of the polymer meshes may releasethe one or more types of entrapped biomolecules. The biomolecules may bedifferent.

In some cases, two or more polymer meshes can be incorporated into theIPN, and the IPN may be tuned for selective release of two biomoleculetypes. In one non-limiting example, an IPN may comprise two polymermeshes that are dissociable via two different mechanisms. A cell beadmay comprise a cell encapsulated in said IPN. During cell lysis, the IPNmay prevent biomolecules of interest (e.g., RNA, proteins) from exitingthe cell bead. During co-partitioning of the cell bead with a gel bead,a first dissociation mechanism may be introduced that dissociates one ofthe polymer meshes (e.g., reduction of disulfide bonds or oxidation ofdihydroxybutane bonds). This dissociation mechanism may increase thepore size of the IPN, thereby allowing biomolecules of interest (e.g.,RNA) to exit out of the cell bead while retaining larger biomolecules ofinterest (e.g., DNA). In some cases, the dissolution may lead to releaseof biomolecules of interest without increasing the bead pore size. Asecond dissociation mechanism may be introduced upon a secondco-partitioning that may allow a second type of nucleic acid (e.g., DNA)to be released. In some cases the second dissociation mechanismdissociates the cell bead, thus allowing for larger biomolecules (e.g.,DNA) to exit the cell bead into the partition.

Systems and Methods for Sample Compartmentalization

In an aspect, the systems and methods described herein provide for thecompartmentalization, depositing, or partitioning of one or moreparticles (e.g., biological particles, macromolecular constituents ofbiological particles, beads, reagents, etc.) into discrete compartmentsor partitions (referred to interchangeably herein as partitions), whereeach partition maintains separation of its own contents from thecontents of other partitions. The partition can be a droplet in anemulsion. A partition may comprise one or more other partitions.

A partition may include one or more particles. A partition may includeone or more types of particles. For example, a partition of the presentdisclosure may comprise one or more biological particles and/ormacromolecular constituents thereof. A partition may comprise one ormore gel beads. A partition may comprise one or more cell beads. Apartition may include a single gel bead, a single cell bead, or both asingle cell bead and single gel bead. A partition may include one ormore reagents. Alternatively, a partition may be unoccupied. Forexample, a partition may not comprise a bead. A cell bead can be abiological particle and/or one or more of its macromolecularconstituents encased inside of a gel or polymer matrix, such as viapolymerization of a droplet containing the biological particle andprecursors capable of being polymerized or gelled. Unique identifiers,such as barcodes, may be injected into the droplets previous to,subsequent to, or concurrently with droplet generation, such as via amicrocapsule (e.g., bead), as described elsewhere herein. Microfluidicchannel networks (e.g., on a chip) can be utilized to generatepartitions as described herein. Alternative mechanisms may also beemployed in the partitioning of individual biological particles,including porous membranes through which aqueous mixtures of cells areextruded into non-aqueous fluids.

The partitions can be flowed within fluid streams. The partitions maycomprise, for example, micro-vesicles that have an outer barriersurrounding an inner fluid center or core. In some cases, the partitionsmay comprise a porous matrix that is capable of entraining and/orretaining materials within its matrix. The partitions can be droplets ofa first phase within a second phase, wherein the first and second phasesare immiscible. For example, the partitions can be droplets of aqueousfluid within a non-aqueous continuous phase (e.g., oil phase). Inanother example, the partitions can be droplets of a non-aqueous fluidwithin an aqueous phase. In some examples, the partitions may beprovided in a water-in-oil emulsion or oil-in-water emulsion. A varietyof different vessels are described in, for example, U.S. Pat.Application Publication No. 2014/0155295, which is entirely incorporatedherein by reference for all purposes. Emulsion systems for creatingstable droplets in non-aqueous or oil continuous phases are describedin, for example, U.S. Pat. Application Publication No. 2010/0105112,which is entirely incorporated herein by reference for all purposes.

In the case of droplets in an emulsion, allocating individual particlesto discrete partitions may in one non-limiting example be accomplishedby introducing a flowing stream of particles in an aqueous fluid into aflowing stream of a non-aqueous fluid, such that droplets are generatedat the junction of the two streams. Fluid properties (e.g., fluid flowrates, fluid viscosities, etc.), particle properties (e.g., volumefraction, particle size, particle concentration, etc.), microfluidicarchitectures (e.g., channel geometry, etc.), and other parameters maybe adjusted to control the occupancy of the resulting partitions (e.g.,number of biological particles per partition, number of beads perpartition, etc.). For example, partition occupancy can be controlled byproviding the aqueous stream at a certain concentration and/or flow rateof particles. To generate single biological particle partitions, therelative flow rates of the immiscible fluids can be selected such that,on average, the partitions may contain less than one biological particleper partition in order to ensure that those partitions that are occupiedare primarily singly occupied. In some cases, partitions among aplurality of partitions may contain at most one biological particle(e.g., bead, DNA, cell or cellular material). In some embodiments, thevarious parameters (e.g., fluid properties, particle properties,microfluidic architectures, etc.) may be selected or adjusted such thata majority of partitions are occupied, for example, allowing for only asmall percentage of unoccupied partitions. The flows and channelarchitectures can be controlled as to ensure a given number of singlyoccupied partitions, less than a certain level of unoccupied partitionsand/or less than a certain level of multiply occupied partitions.

FIG. 1 shows an example of a microfluidic channel structure 100 forpartitioning individual biological particles. The channel structure 100can include channel segments 102, 104, 106 and 108 communicating at achannel junction 110. In operation, a first aqueous fluid 112 thatincludes suspended biological particles (or cells) 114 may betransported along channel segment 102 into junction 110, while a secondfluid 116 that is immiscible with the aqueous fluid 112 is delivered tothe junction 110 from each of channel segments 104 and 106 to creatediscrete droplets 118, 120 of the first aqueous fluid 112 flowing intochannel segment 108, and flowing away from junction 110. The channelsegment 108 may be fluidically coupled to an outlet reservoir where thediscrete droplets can be stored and/or harvested. A discrete dropletgenerated may include an individual biological particle 114 (such asdroplets 118). A discrete droplet generated may include more than oneindividual biological particle 114 (not shown in FIG. 1 ). A discretedroplet may contain no biological particle 114 (such as droplet 120).Each discrete partition may maintain separation of its own contents(e.g., individual biological particle 114) from the contents of otherpartitions.

The second fluid 116 can comprise an oil, such as a fluorinated oil,that includes a fluorosurfactant for stabilizing the resulting droplets,for example, inhibiting subsequent coalescence of the resulting droplets118, 120. Examples of particularly useful partitioning fluids andfluorosurfactants are described, for example, in U.S. Pat. ApplicationPublication No. 2010/0105112, which is entirely incorporated herein byreference for all purposes.

As will be appreciated, the channel segments described herein may becoupled to any of a variety of different fluid sources or receivingcomponents, including reservoirs, tubing, manifolds, or fluidiccomponents of other systems. As will be appreciated, the microfluidicchannel structure 100 may have other geometries. For example, amicrofluidic channel structure can have more than one channel junction.For example, a microfluidic channel structure can have 2, 3, 4, or 5channel segments each carrying particles (e.g., biological particles,cell beads, and/or gel beads) that meet at a channel junction. Fluid maybe directed to flow along one or more channels or reservoirs via one ormore fluid flow units. A fluid flow unit can comprise compressors (e.g.,providing positive pressure), pumps (e.g., providing negative pressure),actuators, and the like to control flow of the fluid. Fluid may also orotherwise be controlled via applied pressure differentials, centrifugalforce, electrokinetic pumping, vacuum, capillary or gravity flow, or thelike.

The generated droplets may comprise two subsets of droplets: (1)occupied droplets 118, containing one or more biological particles 114,and (2) unoccupied droplets 120, not containing any biological particles114. Occupied droplets 118 may comprise singly occupied droplets (havingone biological particle) and multiply occupied droplets (having morethan one biological particle). As described elsewhere herein, in somecases, the majority of occupied partitions can include no more than onebiological particle per occupied partition and some of the generatedpartitions can be unoccupied (of any biological particle). In somecases, though, some of the occupied partitions may include more than onebiological particle. In some cases, the partitioning process may becontrolled such that fewer than about 25% of the occupied partitionscontain more than one biological particle, and in many cases, fewer thanabout 20% of the occupied partitions have more than one biologicalparticle, while in some cases, fewer than about 10% or even fewer thanabout 5% of the occupied partitions include more than one biologicalparticle per partition.

In some cases, it may be desirable to minimize the creation of excessivenumbers of empty partitions, such as to reduce costs and/or increaseefficiency. While this minimization may be achieved by providing asufficient number of biological particles (e.g., biological particles114) at the partitioning junction 110, such as to ensure that at leastone biological particle is encapsulated in a partition, the Poissoniandistribution may expectedly increase the number of partitions thatinclude multiple biological particles. As such, where singly occupiedpartitions are to be obtained, at most about 95%, 90%, 85%, 80%, 75%,70%, 65%, 60%, 55%, 50%, 45%, 40%, 35%, 30%, 25%, 20%, 15%, 10%, 5% orless of the generated partitions can be unoccupied.

In some cases, the flow of one or more of the biological particles(e.g., in channel segment 102), or other fluids directed into thepartitioning junction (e.g., in channel segments 104, 106) can becontrolled such that, in many cases, no more than about 50% of thegenerated partitions, no more than about 25% of the generatedpartitions, or no more than about 10% of the generated partitions areunoccupied. These flows can be controlled so as to present anon-Poissonian distribution of single-occupied partitions whileproviding lower levels of unoccupied partitions. The above noted rangesof unoccupied partitions can be achieved while still providing any ofthe single occupancy rates described above. For example, in many cases,the use of the systems and methods described herein can create resultingpartitions that have multiple occupancy rates of less than about 25%,less than about 20%, less than about 15%, less than about 10%, and inmany cases, less than about 5%, while having unoccupied partitions ofless than about 50%, less than about 40%, less than about 30%, less thanabout 20%, less than about 10%, less than about 5%, or less.

As will be appreciated, the above-described occupancy rates are alsoapplicable to partitions that include both biological particles andadditional reagents, including, but not limited to, microcapsules orbeads (e.g., gel beads) carrying barcoded nucleic acid molecules (e.g.,oligonucleotides) (described in relation to FIG. 2 ). The occupiedpartitions (e.g., at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%,90%, 95%, or 99% of the occupied partitions) can include both amicrocapsule (e.g., bead) comprising barcoded nucleic acid molecules anda biological particle.

In another aspect, in addition to or as an alternative to droplet basedpartitioning, biological particles may be encapsulated within amicrocapsule that comprises an outer shell, layer or porous matrix inwhich is entrained one or more individual biological particles or smallgroups of biological particles. The microcapsule may include otherreagents. Encapsulation of biological particles may be performed by avariety of processes. Such processes may combine an aqueous fluidcontaining the biological particles with a polymeric precursor materialthat may be capable of being formed into a gel or other solid orsemi-solid matrix upon application of a particular stimulus to thepolymer precursor. Such stimuli can include, for example, thermalstimuli (e.g., either heating or cooling), photo-stimuli (e.g., throughphoto-curing), chemical stimuli (e.g., through crosslinking,polymerization initiation of the precursor (e.g., through addedinitiators)), mechanical stimuli, or a combination thereof.

Preparation of microcapsules comprising biological particles may beperformed by a variety of methods. For example, air knife droplet oraerosol generators may be used to dispense droplets of precursor fluidsinto gelling solutions in order to form microcapsules that includeindividual biological particles or small groups of biological particles.Likewise, membrane based encapsulation systems may be used to generatemicrocapsules comprising encapsulated biological particles as describedherein. Microfluidic systems of the present disclosure, such as thatshown in FIG. 1 , may be readily used in encapsulating cells asdescribed herein. In particular, and with reference to FIG. 1 , theaqueous fluid 112 comprising (i) the biological particles 114 and (ii)the polymer precursor material (not shown) is flowed into channeljunction 110, where it is partitioned into droplets 118, 120 through theflow of non-aqueous fluid 116. In the case of encapsulation methods,non-aqueous fluid 116 may also include an initiator (not shown) to causepolymerization and/or crosslinking of the polymer precursor to form themicrocapsule that includes the entrained biological particles. Examplesof polymer precursor/initiator pairs include those described in U.S.Pat. Application Publication No. 2014/0378345, which is entirelyincorporated herein by reference for all purposes.

For example, in the case where the polymer precursor material comprisesa linear polymer material, such as a linear polyacrylamide, PEG, orother linear polymeric material, the activation agent may comprise across-linking agent, or a chemical that activates a cross-linking agentwithin the formed droplets. Likewise, for polymer precursors thatcomprise polymerizable monomers, the activation agent may comprise apolymerization initiator. For example, in certain cases, where thepolymer precursor comprises a mixture of acrylamide monomer with aN,N′-bis-(acryloyl)cystamine (BAC) comonomer, agents such as ammoniumpersulfate (APS) and tetraethylmethylenediamine (TEMED) and may beprovided within the second fluid streams 116 in channel segments 104 and106, which can initiate and catalyze the copolymerization of theacrylamide and BAC into a cross-linked polymer network, or hydrogel.Other non-limiting examples of initiators include azide-based reagents(e.g., VA-086) and lithium phenyl-trimethylbenzoylphosphinate.

Upon contact of the second fluid stream 116 with the first fluid stream112 at junction 110, during formation of droplets, the TEMED may diffusefrom the second fluid 116 into the aqueous fluid 112 comprising thelinear polyacrylamide, which will activate the crosslinking of thepolyacrylamide within the droplets 118, 120, resulting in the formationof gel (e.g., hydrogel) microcapsules, as solid or semi-solid beads orparticles entraining the cells 114. Although described in terms ofpolyacrylamide encapsulation, other ‘activatable’ encapsulationcompositions may also be employed in the context of the methods andcompositions described herein. For example, formation of alginatedroplets followed by exposure to divalent metal ions (e.g., Ca²⁺ ions),can be used as an encapsulation process using the described processes.Likewise, agarose droplets may also be transformed into capsules throughtemperature based gelling (e.g., upon cooling, etc.). In anotherexample, addition of a complementary nucleic acid (e.g., DNA) may beused to crosslink or un-crosslink nucleic acid molecules that areconjugated to a polymer network.

In some cases, encapsulated biological particles can be selectivelyreleasable from the microcapsule, such as through passage of time orupon application of a particular stimulus, that degrades themicrocapsule sufficiently to allow the biological particles (e.g.,cell), or its other contents to be released from the microcapsule, suchas into a partition (e.g., droplet). For example, in the case of thepolyacrylamide polymer described above, degradation of the microcapsulemay be accomplished through the introduction of an appropriate reducingagent, such as DTT or the like, to cleave disulfide bonds thatcross-link the polymer matrix. See, for example, U.S. Pat. ApplicationPublication No. 2014/0378345, which is entirely incorporated herein byreference for all purposes.

The biological particle can be subjected to other conditions sufficientto polymerize or gel the precursors. The conditions sufficient topolymerize or gel the precursors may comprise exposure to heating,cooling, electromagnetic radiation, and/or light. The conditionssufficient to polymerize or gel the precursors may comprise anyconditions sufficient to polymerize or gel the precursors. Followingpolymerization or gelling, a polymer or gel may be formed around thebiological particle. The polymer or gel may be diffusively permeable tochemical or biochemical reagents. The polymer or gel may be diffusivelyimpermeable to macromolecular constituents of the biological particle.In this manner, the polymer or gel may act to allow the biologicalparticle to be subjected to chemical or biochemical operations whilespatially confining the macromolecular constituents to a region of thedroplet defined by the polymer or gel. The polymer or gel may includeone or more of disulfide cross-linked polyacrylamide, agarose, alginate,polyvinyl alcohol, polyethylene glycol (PEG)-diacrylate, PEG-acrylate,PEG-thiol, PEG-azide, PEG-alkyne, other acrylates, chitosan, hyaluronicacid, collagen, fibrin, gelatin, or elastin. The polymer or gel maycomprise any other polymer or gel.

The polymer or gel may be functionalized to bind to targeted analytes,such as nucleic acids, proteins, carbohydrates, lipids or otheranalytes. The polymer or gel may be polymerized or gelled via a passivemechanism. The polymer or gel may be stable in alkaline or acidicconditions or at elevated temperature. The polymer or gel may havemechanical properties similar to the mechanical properties of the bead.For instance, the polymer or gel may be of a similar size to the bead.The polymer or gel may have a mechanical strength (e.g., tensilestrength, compressive strength, stiffness, toughness, etc.) similar tothat of the bead. The polymer or gel may be of a lower density than anoil. The polymer or gel may be of a density that is roughly similar tothat of a buffer. The polymer or gel may have a tunable pore size. Thepore size may be chosen to, for instance, retain denatured nucleicacids. The pore size may be chosen to maintain diffusive permeability toexogenous chemicals such as sodium hydroxide (NaOH) and/or endogenouschemicals such as inhibitors. The polymer or gel may be biocompatible.The polymer or gel may maintain or enhance cell viability. The polymeror gel may be biochemically compatible. The polymer or gel may bepolymerized and/or depolymerized thermally, chemically, enzymatically,and/or optically.

The polymer may comprise poly(acrylamide-co-acrylic acid) crosslinkedwith disulfide linkages. The preparation of the polymer may comprise atwo-step reaction. In the first activation step,poly(acrylamide-co-acrylic acid) may be exposed to an acylating agent toconvert carboxylic acids to esters. For instance, thepoly(acrylamide-co-acrylic acid) may be exposed to4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium chloride(DMTMM). The polyacrylamide-co-acrylic acid may be exposed to othersalts of 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium. Inthe second cross-linking step, the ester formed in the first step may beexposed to a disulfide crosslinking agent. For instance, the ester maybe exposed to cystamine (2,2′-dithiobis(ethylamine)). Following the twosteps, the biological particle may be surrounded by polyacrylamidestrands linked together by disulfide bridges. In this manner, thebiological particle may be encased inside of or comprise a gel or matrix(e.g., polymer matrix) to form a “cell bead.” A cell bead can containbiological particles (e.g., a cell) or macromolecular constituents(e.g., RNA, DNA, proteins, etc.) of biological particles, as describedelsewhere herein.

Encapsulated biological particles can provide certain potentialadvantages of being more storable and more portable than droplet-basedpartitioned biological particles. Furthermore, in some cases, it may bedesirable to allow biological particles to incubate for a select periodof time before analysis, such as in order to characterize changes insuch biological particles over time, either in the presence or absenceof different stimuli. In such cases, encapsulation may allow for longerincubation than partitioning in emulsion droplets, although in somecases, droplet partitioned biological particles may also be incubatedfor different periods of time, e.g., at least 10 seconds, at least 30seconds, at least 1 minute, at least 5 minutes, at least 10 minutes, atleast 30 minutes, at least 1 hour, at least 2 hours, at least 5 hours,or at least 10 hours or more. The encapsulation of biological particlesmay constitute the partitioning of the biological particles into whichother reagents are co-partitioned. Alternatively or in addition,encapsulated biological particles may be readily deposited into otherpartitions (e.g., droplets) as described above.

Beads

A partition may comprise one or more unique identifiers, such asbarcodes. Barcodes may be previously, subsequently or concurrentlydelivered to the partitions that hold the compartmentalized orpartitioned biological particle. For example, barcodes may be injectedinto droplets previous to, subsequent to, or concurrently with dropletgeneration. The delivery of the barcodes to a particular partitionallows for the later attribution of the characteristics of theindividual biological particle to the particular partition. Barcodes maybe delivered, for example on a nucleic acid molecule (e.g., anoligonucleotide), to a partition via any suitable mechanism. Barcodednucleic acid molecules can be delivered to a partition via amicrocapsule. A microcapsule, in some instances, can comprise a bead.Beads are described in further detail below.

In some cases, barcoded nucleic acid molecules can be initiallyassociated with the microcapsule and then released from themicrocapsule. Release of the barcoded nucleic acid molecules can bepassive (e.g., by diffusion out of the microcapsule). In addition oralternatively, release from the microcapsule can be upon application ofa stimulus which allows the barcoded nucleic acid nucleic acid moleculesto dissociate or to be released from the microcapsule. Such stimulus maydisrupt the microcapsule, an interaction that couples the barcodednucleic acid molecules to or within the microcapsule, or both. Suchstimulus can include, for example, a thermal stimulus, photo-stimulus,chemical stimulus (e.g., change in pH or use of a reducing agent(s)), amechanical stimulus, a radiation stimulus; a biological stimulus (e.g.,enzyme), or any combination thereof.

FIG. 2 shows an example of a microfluidic channel structure 200 fordelivering barcode carrying beads to droplets. The channel structure 200can include channel segments 201, 202, 204, 206 and 208 communicating ata channel junction 210. In operation, the channel segment 201 maytransport an aqueous fluid 212 that includes a plurality of beads 214(e.g., with nucleic acid molecules, oligonucleotides, molecular tags)along the channel segment 201 into junction 210. The plurality of beads214 may be sourced from a suspension of beads. For example, the channelsegment 201 may be connected to a reservoir comprising an aqueoussuspension of beads 214. The channel segment 202 may transport theaqueous fluid 212 that includes a plurality of biological particles 216along the channel segment 202 into junction 210. The plurality ofbiological particles 216 may be sourced from a suspension of biologicalparticles. For example, the channel segment 202 may be connected to areservoir comprising an aqueous suspension of biological particles 216.In some instances, the aqueous fluid 212 in either the first channelsegment 201 or the second channel segment 202, or in both segments, caninclude one or more reagents, as further described below. A second fluid218 that is immiscible with the aqueous fluid 212 (e.g., oil) can bedelivered to the junction 210 from each of channel segments 204 and 206.Upon meeting of the aqueous fluid 212 from each of channel segments 201and 202 and the second fluid 218 from each of channel segments 204 and206 at the channel junction 210, the aqueous fluid 212 can bepartitioned as discrete droplets 220 in the second fluid 218 and flowaway from the junction 210 along channel segment 208. The channelsegment 208 may deliver the discrete droplets to an outlet reservoirfluidly coupled to the channel segment 208, where they may be harvested.

As an alternative, the channel segments 201 and 202 may meet at anotherjunction upstream of the junction 210. At such junction, beads andbiological particles may form a mixture that is directed along anotherchannel to the junction 210 to yield droplets 220. The mixture mayprovide the beads and biological particles in an alternating fashion,such that, for example, a droplet comprises a single bead and a singlebiological particle.

Beads, biological particles and droplets may flow along channels atsubstantially regular flow profiles (e.g., at regular flow rates). Suchregular flow profiles may permit a droplet to include a single bead anda single biological particle. Such regular flow profiles may permit thedroplets to have an occupancy (e.g., droplets having beads andbiological particles) greater than 5%, 10%, 20%, 30%, 40%, 50%, 60%,70%, 80%, 90%, or 95%. Such regular flow profiles and devices that maybe used to provide such regular flow profiles are provided in, forexample, U.S. Pat. Publication No. 2015/0292988, which is entirelyincorporated herein by reference.

The second fluid 218 can comprise an oil, such as a fluorinated oil,that includes a fluorosurfactant for stabilizing the resulting droplets,for example, inhibiting subsequent coalescence of the resulting droplets220. Other surfactants such as Span80, Triton X-100, SDS,perfluorooctanol (PFO), perfluoropolyethers, etc. may also be employedto prevent coalescence of droplets.

A discrete droplet that is generated may include an individualbiological particle 216. A discrete droplet that is generated mayinclude a barcode or other reagent carrying bead 214. A discrete dropletgenerated may include both an individual biological particle and abarcode carrying bead, such as droplets 220. In some instances, adiscrete droplet may include more than one individual biologicalparticle or no biological particle. In some instances, a discretedroplet may include more than one bead or no bead. A discrete dropletmay be unoccupied (e.g., no beads, no biological particles).

Beneficially, a discrete droplet partitioning a biological particle anda barcode carrying bead may effectively allow the attribution of thebarcode to macromolecular constituents of the biological particle withinthe partition. The contents of a partition may remain discrete from thecontents of other partitions.

As will be appreciated, the channel segments described herein may becoupled to any of a variety of different fluid sources or receivingcomponents, including reservoirs, tubing, manifolds, or fluidiccomponents of other systems. As will be appreciated, the microfluidicchannel structure 200 may have other geometries. For example, amicrofluidic channel structure can have more than one channel junctions.For example, a microfluidic channel structure can have 2, 3, 4, or 5channel segments each carrying beads that meet at a channel junction.Fluid may be directed flow along one or more channels or reservoirs viaone or more fluid flow units. A fluid flow unit can comprise compressors(e.g., providing positive pressure), pumps (e.g., providing negativepressure), actuators, and the like to control flow of the fluid. Fluidmay also or otherwise be controlled via applied pressure differentials,centrifugal force, electrokinetic pumping, vacuum, capillary or gravityflow, or the like.

A bead may be porous, non-porous, solid, semi-solid, semi-fluidic,fluidic, and/or a combination thereof. In some instances, a bead may bedissolvable, disruptable, and/or degradable. In some cases, a bead maynot be degradable. In some cases, the bead may be a gel bead. A gel beadmay be a hydrogel bead. A gel bead may be formed from molecularprecursors, such as a polymeric or monomeric species. A semi-solid beadmay be a liposomal bead. Solid beads may comprise metals including ironoxide, gold, and silver. In some cases, the bead may be a silica bead.In some cases, the bead can be rigid. In other cases, the bead may beflexible and/or compressible.

A bead may be of any suitable shape. Examples of bead shapes include,but are not limited to, spherical, non-spherical, oval, oblong,amorphous, circular, cylindrical, and variations thereof.

Beads may be of uniform size or heterogeneous size. In some cases, thediameter of a bead may be at least about 10 nanometers (nm), 100 nm, 500nm, 1 micrometer (µm), 5 µm, 10 µm, 20 µm, 30 µm, 40 µm, 50 µm, 60 µm,70 µm, 80 µm, 90 µm, 100 µm, 250 µm, 500 µm, 1 mm, or greater. In somecases, a bead may have a diameter of less than about 10 nm, 100 nm, 500nm, 1 µm, 5 µm, 10 µm, 20 µm, 30 µm, 40 µm, 50 µm, 60 µm, 70 µm, 80 µm,90 µm, 100 µm, 250 µm, 500 µm, 1 mm, or less. In some cases, a bead mayhave a diameter in the range of about 40-75 µm, 30-75 µm, 20-75 µm,40-85 µm, 40-95 µm, 20-100 µm, 10-100 µm, 1-100 µm, 20-250 µm, or 20-500µm.

In certain aspects, beads can be provided as a population or pluralityof beads having a relatively monodisperse size distribution. Where itmay be desirable to provide relatively consistent amounts of reagentswithin partitions, maintaining relatively consistent beadcharacteristics, such as size, can contribute to the overallconsistency. In particular, the beads described herein may have sizedistributions that have a coefficient of variation in theircross-sectional dimensions of less than 50%, less than 40%, less than30%, less than 20%, and in some cases less than 15%, less than 10%, lessthan 5%, or less.

A bead may comprise natural and/or synthetic materials. For example, abead can comprise a natural polymer, a synthetic polymer or both naturaland synthetic polymers. Examples of natural polymers include proteinsand sugars such as deoxyribonucleic acid, rubber, cellulose, starch(e.g., amylose, amylopectin), proteins, enzymes, polysaccharides, silks,polyhydroxyalkanoates, chitosan, dextran, collagen, carrageenan,ispaghula, acacia, agar, gelatin, shellac, sterculia gum, xanthan gum,Corn sugar gum, guar gum, gum karaya, agarose, alginic acid, alginate,or natural polymers thereof. Examples of synthetic polymers includeacrylics, nylons, silicones, spandex, viscose rayon, polycarboxylicacids, polyvinyl acetate, polyacrylamide, polyacrylate, polyethyleneglycol, polyurethanes, polylactic acid, silica, polystyrene,polyacrylonitrile, polybutadiene, polycarbonate, polyethylene,polyethylene terephthalate, poly(chlorotrifluoroethylene), poly(ethyleneoxide), poly(ethylene terephthalate), polyethylene, polyisobutylene,poly(methyl methacrylate), poly(oxymethylene), polyformaldehyde,polypropylene, polystyrene, poly(tetrafluoroethylene), poly(vinylacetate), poly(vinyl alcohol), poly(vinyl chloride), poly(vinylidenedichloride), poly(vinylidene difluoride), poly(vinyl fluoride) and/orcombinations (e.g., co-polymers) thereof. Beads may also be formed frommaterials other than polymers, including lipids, micelles, liposomes,ceramics, glass-ceramics, material composites, metals, other inorganicmaterials, and others.

In some instances, the bead may contain molecular precursors (e.g.,monomers or polymers), which may form a polymer network viapolymerization of the molecular precursors. In some cases, a precursormay be an already polymerized species capable of undergoing furtherpolymerization via, for example, a chemical cross-linkage. In somecases, a precursor can comprise one or more of an acrylamide or amethacrylamide monomer, oligomer, or polymer. In some cases, the beadmay comprise prepolymers, which are oligomers capable of furtherpolymerization. For example, polyurethane beads may be prepared usingprepolymers. In some cases, the bead may contain individual polymersthat may be further polymerized together. In some cases, beads may begenerated via polymerization of different precursors, such that theycomprise mixed polymers, co-polymers, and/or block co-polymers. In somecases, the bead may comprise covalent or ionic bonds between polymericprecursors (e.g., monomers, oligomers, linear polymers), nucleic acidmolecules (e.g., oligonucleotides), primers, and other entities. In somecases, the covalent bonds can be carbon-carbon bonds, thioether bonds,or carbon-heteroatom bonds.

Cross-linking may be permanent or reversible, depending upon theparticular cross-linker used. Reversible cross-linking may allow for thepolymer to linearize or dissociate under appropriate conditions. In somecases, reversible cross-linking may also allow for reversible attachmentof a material bound to the surface of a bead. In some cases, across-linker may form disulfide linkages. In some cases, the chemicalcross-linker forming disulfide linkages may be cystamine or a modifiedcystamine.

In some cases, disulfide linkages can be formed between molecularprecursor units (e.g., monomers, oligomers, or linear polymers) orprecursors incorporated into a bead and nucleic acid molecules (e.g.,oligonucleotides). Cystamine (including modified cystamines), forexample, is an organic agent comprising a disulfide bond that may beused as a crosslinker agent between individual monomeric or polymericprecursors of a bead. Polyacrylamide may be polymerized in the presenceof cystamine or a species comprising cystamine (e.g., a modifiedcystamine) to generate polyacrylamide gel beads comprising disulfidelinkages (e.g., chemically degradable beads comprisingchemically-reducible cross-linkers). The disulfide linkages may permitthe bead to be degraded (or dissolved) upon exposure of the bead to areducing agent.

In some cases, chitosan, a linear polysaccharide polymer, may becrosslinked with glutaraldehyde via hydrophilic chains to form a bead.Crosslinking of chitosan polymers may be achieved by chemical reactionsthat are initiated by heat, pressure, change in pH, and/or radiation.

In some cases, a bead may comprise an acrydite moiety, which in certainaspects may be used to attach one or more nucleic acid molecules (e.g.,barcode sequence, barcoded nucleic acid molecule, barcodedoligonucleotide, primer, or other oligonucleotide) to the bead. In somecases, an acrydite moiety can refer to an acrydite analogue generatedfrom the reaction of acrydite with one or more species, such as, thereaction of acrydite with other monomers and cross-linkers during apolymerization reaction. Acrydite moieties may be modified to formchemical bonds with a species to be attached, such as a nucleic acidmolecule (e.g., barcode sequence, barcoded nucleic acid molecule,barcoded oligonucleotide, primer, or other oligonucleotide). Acryditemoieties may be modified with thiol groups capable of forming adisulfide bond or may be modified with groups already comprising adisulfide bond. The thiol or disulfide (via disulfide exchange) may beused as an anchor point for a species to be attached or another part ofthe acrydite moiety may be used for attachment. In some cases,attachment can be reversible, such that when the disulfide bond isbroken (e.g., in the presence of a reducing agent), the attached speciesis released from the bead. In other cases, an acrydite moiety cancomprise a reactive hydroxyl group that may be used for attachment.

Functionalization of beads for attachment of nucleic acid molecules(e.g., oligonucleotides) may be achieved through a wide range ofdifferent approaches, including activation of chemical groups within apolymer, incorporation of active or activatable functional groups in thepolymer structure, or attachment at the pre-polymer or monomer stage inbead production.

For example, precursors (e.g., monomers, cross-linkers) that arepolymerized to form a bead may comprise acrydite moieties, such thatwhen a bead is generated, the bead also comprises acrydite moieties. Theacrydite moieties can be attached to a nucleic acid molecule (e.g.,oligonucleotide), which may include a priming sequence (e.g., a primerfor amplifying target nucleic acids, random primer, primer sequence formRNA) and/or one or more barcode sequences. The one or more barcodesequences may include sequences that are the same for all nucleic acidmolecules coupled to a given bead and/or sequences that are differentacross all nucleic acid molecules coupled to the given bead. The nucleicacid molecule may be incorporated into the bead.

In some cases, the nucleic acid molecule can comprise a functionalsequence, for example, for attachment to a sequencing flow cell, suchas, for example, a P5 sequence for Illumina® sequencing. In some cases,the nucleic acid molecule or derivative thereof (e.g., oligonucleotideor polynucleotide generated from the nucleic acid molecule) can compriseanother functional sequence, such as, for example, a P7 sequence forattachment to a sequencing flow cell for Illumina sequencing. In somecases, the nucleic acid molecule can comprise a barcode sequence. Insome cases, the primer can further comprise a unique molecularidentifier (UMI). In some cases, the primer can comprise an R1 primersequence for Illumina sequencing. In some cases, the primer can comprisean R2 primer sequence for Illumina sequencing. Examples of such nucleicacid molecules (e.g., oligonucleotides, polynucleotides, etc.) and usesthereof, as may be used with compositions, devices, methods and systemsof the present disclosure, are provided in U.S. Pat. Pub. Nos.2014/0378345 and 2015/0376609, each of which is entirely incorporatedherein by reference.

FIG. 8 illustrates an example of a barcode carrying bead. A nucleic acidmolecule 802, such as an oligonucleotide, can be coupled to a bead 804by a releasable linkage 806, such as, for example, a disulfide linker.The same bead 804 may be coupled (e.g., via releasable linkage) to oneor more other nucleic acid molecules 818, 820. The nucleic acid molecule802 may be or comprise a barcode. As noted elsewhere herein, thestructure of the barcode may comprise a number of sequence elements. Thenucleic acid molecule 802 may comprise a functional sequence 808 thatmay be used in subsequent processing. For example, the functionalsequence 808 may include one or more of a sequencer specific flow cellattachment sequence (e.g., a P5 sequence for Illumina® sequencingsystems) and a sequencing primer sequence (e.g., a R1 primer forIllumina® sequencing systems). The nucleic acid molecule 802 maycomprise a barcode sequence 810 for use in barcoding the sample (e.g.,DNA, RNA, protein, etc.). In some cases, the barcode sequence 810 can bebead-specific such that the barcode sequence 810 is common to allnucleic acid molecules (e.g., including nucleic acid molecule 802)coupled to the same bead 804. Alternatively or in addition, the barcodesequence 810 can be partition-specific such that the barcode sequence810 is common to all nucleic acid molecules coupled to one or more beadsthat are partitioned into the same partition. The nucleic acid molecule802 may comprise a specific priming sequence 812, such as an mRNAspecific priming sequence (e.g., poly-T sequence), a targeted primingsequence, and/or a random priming sequence. The nucleic acid molecule802 may comprise an anchoring sequence 814 to ensure that the specificpriming sequence 812 hybridizes at the sequence end (e.g., of the mRNA).For example, the anchoring sequence 814 can include a random shortsequence of nucleotides, such as a 1-mer, 2-mer, 3-mer or longersequence, which can ensure that a poly-T segment is more likely tohybridize at the sequence end of the poly-A tail of the mRNA.

The nucleic acid molecule 802 may comprise a unique molecularidentifying sequence 816 (e.g., unique molecular identifier (UMI)). Insome cases, the unique molecular identifying sequence 816 may comprisefrom about 5 to about 8 nucleotides. Alternatively, the unique molecularidentifying sequence 816 may compress less than about 5 or more thanabout 8 nucleotides. The unique molecular identifying sequence 816 maybe a unique sequence that varies across individual nucleic acidmolecules (e.g., 802, 818, 820, etc.) coupled to a single bead (e.g.,bead 804). In some cases, the unique molecular identifying sequence 816may be a random sequence (e.g., such as a random N-mer sequence). Forexample, the UMI may provide a unique identifier of the starting mRNAmolecule that was captured, in order to allow quantitation of the numberof original expressed RNA. As will be appreciated, although FIG. 8 showsthree nucleic acid molecules 802, 818, 820 coupled to the surface of thebead 804, an individual bead may be coupled to any number of individualnucleic acid molecules, for example, from one to tens to hundreds ofthousands or even millions of individual nucleic acid molecules. Therespective barcodes for the individual nucleic acid molecules cancomprise both common sequence segments or relatively common sequencesegments (e.g., 808, 810, 812, etc.) and variable or unique sequencesegments (e.g., 816) between different individual nucleic acid moleculescoupled to the same bead.

In operation, a biological particle (e.g., cell, DNA, RNA, etc.) can beco-partitioned along with a barcode bearing bead 804. The barcodednucleic acid molecules 802, 818, 820 can be released from the bead 804in the partition. By way of example, in the context of analyzing sampleRNA, the poly-T segment (e.g., 812) of one of the released nucleic acidmolecules (e.g., 802) can hybridize to the poly-A tail of a mRNAmolecule. Reverse transcription may result in a cDNA transcript of themRNA, but which transcript includes each of the sequence segments 808,810, 816 of the nucleic acid molecule 802. Because the nucleic acidmolecule 802 comprises an anchoring sequence 814, it will more likelyhybridize to and prime reverse transcription at the sequence end of thepoly-A tail of the mRNA. Within any given partition, all of the cDNAtranscripts of the individual mRNA molecules may include a commonbarcode sequence segment 810. However, the transcripts made from thedifferent mRNA molecules within a given partition may vary at the uniquemolecular identifying sequence 812 segment (e.g., UMI segment).Beneficially, even following any subsequent amplification of thecontents of a given partition, the number of different UMIs can beindicative of the quantity of mRNA originating from a given partition,and thus from the biological particle (e.g., cell). As noted above, thetranscripts can be amplified, cleaned up and sequenced to identify thesequence of the cDNA transcript of the mRNA, as well as to sequence thebarcode segment and the UMI segment. While a poly-T primer sequence isdescribed, other targeted or random priming sequences may also be usedin priming the reverse transcription reaction. Likewise, althoughdescribed as releasing the barcoded oligonucleotides into the partition,in some cases, the nucleic acid molecules bound to the bead (e.g., gelbead) may be used to hybridize and capture the mRNA on the solid phaseof the bead, for example, in order to facilitate the separation of theRNA from other cell contents.

In some cases, precursors comprising a functional group that is reactiveor capable of being activated such that it becomes reactive can bepolymerized with other precursors to generate gel beads comprising theactivated or activatable functional group. The functional group may thenbe used to attach additional species (e.g., disulfide linkers, primers,other oligonucleotides, etc.) to the gel beads. For example, someprecursors comprising a carboxylic acid (COOH) group can copolymerizewith other precursors to form a gel bead that also comprises a COOHfunctional group. In some cases, acrylic acid (a species comprising freeCOOH groups), acrylamide, and bis(acryloyl)cystamine can beco-polymerized together to generate a gel bead comprising free COOHgroups. The COOH groups of the gel bead can be activated (e.g., via1-Ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) andN-Hydroxysuccinimide (NHS) or4-(4,6-Dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium chloride(DMTMM)) such that they are reactive (e.g., reactive to amine functionalgroups where EDC/NHS or DMTMM are used for activation). The activatedCOOH groups can then react with an appropriate species (e.g., a speciescomprising an amine functional group where the carboxylic acid groupsare activated to be reactive with an amine functional group) comprisinga moiety to be linked to the bead.

Beads comprising disulfide linkages in their polymeric network may befunctionalized with additional species via reduction of some of thedisulfide linkages to free thiols. The disulfide linkages may be reducedvia, for example, the action of a reducing agent (e.g., DTT, TCEP, etc.)to generate free thiol groups, without dissolution of the bead. Freethiols of the beads can then react with free thiols of a species or aspecies comprising another disulfide bond (e.g., via thiol-disulfideexchange) such that the species can be linked to the beads (e.g., via agenerated disulfide bond). In some cases, free thiols of the beads mayreact with any other suitable group. For example, free thiols of thebeads may react with species comprising an acrydite moiety. The freethiol groups of the beads can react with the acrydite via Michaeladdition chemistry, such that the species comprising the acrydite islinked to the bead. In some cases, uncontrolled reactions can beprevented by inclusion of a thiol capping agent such asN-ethylmalieamide or iodoacetate.

Activation of disulfide linkages within a bead can be controlled suchthat only a small number of disulfide linkages are activated. Controlmay be exerted, for example, by controlling the concentration of areducing agent used to generate free thiol groups and/or concentrationof reagents used to form disulfide bonds in bead polymerization. In somecases, a low concentration (e.g., molecules of reducing agent:gel beadratios of less than or equal to about 1:100,000,000,000, less than orequal to about 1: 10,000,000,000, less than or equal to about 1:1,000,000,000, less than or equal to about 1:100,000,000, less than orequal to about 1:10,000,000, less than or equal to about 1:1,000,000,less than or equal to about 1:100,000, less than or equal to about1:10,000) of reducing agent may be used for reduction. Controlling thenumber of disulfide linkages that are reduced to free thiols may beuseful in ensuring bead structural integrity during functionalization.In some cases, optically-active agents, such as fluorescent dyes, may becoupled to beads via free thiol groups of the beads and used to quantifythe number of free thiols present in a bead and/or track a bead.

In some cases, addition of moieties to a gel bead after gel beadformation may be advantageous. For example, addition of anoligonucleotide (e.g., barcoded oligonucleotide) after gel beadformation may avoid loss of the species during chain transfertermination that can occur during polymerization. Moreover, smallerprecursors (e.g., monomers or cross linkers that do not comprise sidechain groups and linked moieties) may be used for polymerization and canbe minimally hindered from growing chain ends due to viscous effects. Insome cases, functionalization after gel bead synthesis can minimizeexposure of species (e.g., oligonucleotides) to be loaded withpotentially damaging agents (e.g., free radicals) and/or chemicalenvironments. In some cases, the generated gel may possess an uppercritical solution temperature (UCST) that can permit temperature drivenswelling and collapse of a bead. Such functionality may aid inoligonucleotide (e.g., a primer) infiltration into the bead duringsubsequent functionalization of the bead with the oligonucleotide.Post-production functionalization may also be useful in controllingloading ratios of species in beads, such that, for example, thevariability in loading ratio is minimized. Species loading may also beperformed in a batch process such that a plurality of beads can befunctionalized with the species in a single batch.

A bead injected or otherwise introduced into a partition may comprisereleasably, cleavably, or reversibly attached barcodes. A bead injectedor otherwise introduced into a partition may comprise activatablebarcodes. A bead injected or otherwise introduced into a partition maybe degradable, disruptable, or dissolvable beads.

Barcodes can be releasably, cleavably or reversibly attached to thebeads such that barcodes can be released or be releasable throughcleavage of a linkage between the barcode molecule and the bead, orreleased through degradation of the underlying bead itself, allowing thebarcodes to be accessed or be accessible by other reagents, or both. Innon-limiting examples, cleavage may be achieved through reduction ofdi-sulfide bonds, use of restriction enzymes, photo-activated cleavage,or cleavage via other types of stimuli (e.g., chemical, thermal, pH,enzymatic, etc.) and/or reactions, such as described elsewhere herein.Releasable barcodes may sometimes be referred to as being activatable,in that they are available for reaction once released. Thus, forexample, an activatable barcode may be activated by releasing thebarcode from a bead (or other suitable type of partition describedherein). Other activatable configurations are also envisioned in thecontext of the described methods and systems.

In addition to, or as an alternative to the cleavable linkages betweenthe beads and the associated molecules, such as barcode containingnucleic acid molecules (e.g., barcoded oligonucleotides), the beads maybe degradable, disruptable, or dissolvable spontaneously or uponexposure to one or more stimuli (e.g., temperature changes, pH changes,exposure to particular chemical species or phase, exposure to light,reducing agent, etc.). In some cases, a bead may be dissolvable, suchthat material components of the beads are solubilized when exposed to aparticular chemical species or an environmental change, such as a changetemperature or a change in pH. In some cases, a gel bead can be degradedor dissolved at elevated temperature and/or in alkaline conditions. Insome cases, a bead may be thermally degradable such that when the beadis exposed to an appropriate change in temperature (e.g., heat), thebead degrades. Degradation or dissolution of a bead bound to a species(e.g., a nucleic acid molecule, e.g., barcoded oligonucleotide) mayresult in release of the species from the bead.

As will be appreciated from the above disclosure, the degradation of abead may refer to the dissociation of a bound or entrained species froma bead, both with and without structurally degrading the physical beaditself. For example, the degradation of the bead may involve cleavage ofa cleavable linkage via one or more species and/or methods describedelsewhere herein. In another example, entrained species may be releasedfrom beads through osmotic pressure differences due to, for example,changing chemical environments. By way of example, alteration of beadpore sizes due to osmotic pressure differences can generally occurwithout structural degradation of the bead itself. In some cases, anincrease in pore size due to osmotic swelling of a bead can permit therelease of entrained species within the bead. In other cases, osmoticshrinking of a bead may cause a bead to better retain an entrainedspecies due to pore size contraction.

A degradable bead may be introduced into a partition, such as a dropletof an emulsion or a well, such that the bead degrades within thepartition and any associated species (e.g., oligonucleotides) arereleased within the droplet when the appropriate stimulus is applied.The free species (e.g., oligonucleotides, nucleic acid molecules) mayinteract with other reagents contained in the partition. For example, apolyacrylamide bead comprising cystamine and linked, via a disulfidebond, to a barcode sequence, may be combined with a reducing agentwithin a droplet of a water-in-oil emulsion. Within the droplet, thereducing agent can break the various disulfide bonds, resulting in beaddegradation and release of the barcode sequence into the aqueous, innerenvironment of the droplet. In another example, heating of a dropletcomprising a bead-bound barcode sequence in basic solution may alsoresult in bead degradation and release of the attached barcode sequenceinto the aqueous, inner environment of the droplet.

Any suitable number of molecular tag molecules (e.g., primer, barcodedoligonucleotide) can be associated with a bead such that, upon releasefrom the bead, the molecular tag molecules (e.g., primer, e.g., barcodedoligonucleotide) are present in the partition at a pre-definedconcentration. Such pre-defined concentration may be selected tofacilitate certain reactions for generating a sequencing library, e.g.,amplification, within the partition. In some cases, the pre-definedconcentration of the primer can be limited by the process of producingnucleic acid molecule (e.g., oligonucleotide) bearing beads.

In some cases, beads can be non-covalently loaded with one or morereagents. The beads can be non-covalently loaded by, for instance,subjecting the beads to conditions sufficient to swell the beads,allowing sufficient time for the reagents to diffuse into the interiorsof the beads, and subjecting the beads to conditions sufficient tode-swell the beads. The swelling of the beads may be accomplished, forinstance, by placing the beads in a thermodynamically favorable solvent,subjecting the beads to a higher or lower temperature, subjecting thebeads to a higher or lower ion concentration, and/or subjecting thebeads to an electric field. The swelling of the beads may beaccomplished by various swelling methods. The de-swelling of the beadsmay be accomplished, for instance, by transferring the beads in athermodynamically unfavorable solvent, subjecting the beads to lower orhigh temperatures, subjecting the beads to a lower or higher ionconcentration, and/or removing an electric field. The de-swelling of thebeads may be accomplished by various de-swelling methods. Transferringthe beads may cause pores in the bead to shrink. The shrinking may thenhinder reagents within the beads from diffusing out of the interiors ofthe beads. The hindrance may be due to steric interactions between thereagents and the interiors of the beads. The transfer may beaccomplished microfluidically. For instance, the transfer may beachieved by moving the beads from one co-flowing solvent stream to adifferent co-flowing solvent stream. The swellability and/or pore sizeof the beads may be adjusted by changing the polymer composition of thebead.

In some cases, an acrydite moiety linked to a precursor, another specieslinked to a precursor, or a precursor itself can comprise a labile bond,such as chemically, thermally, or photosensitive bond e.g., disulfidebond, UV-sensitive bond, or the like. Once acrydite moieties or othermoieties comprising a labile bond are incorporated into a bead, the beadmay also comprise the labile bond. The labile bond may be, for example,useful in reversibly linking (e.g., covalently linking) species (e.g.,barcodes, primers, etc.) to a bead. In some cases, a thermally labilebond may include a nucleic acid hybridization based attachment, e.g.,where an oligonucleotide is hybridized to a complementary sequence thatis attached to the bead, such that thermal melting of the hybridreleases the oligonucleotide, e.g., a barcode containing sequence, fromthe bead or microcapsule.

The addition of multiple types of labile bonds to a gel bead may resultin the generation of a bead capable of responding to varied stimuli.Each type of labile bond may be sensitive to an associated stimulus(e.g., chemical stimulus, light, temperature, enzymatic, etc.) such thatrelease of species attached to a bead via each labile bond may becontrolled by the application of the appropriate stimulus. Suchfunctionality may be useful in controlled release of species from a gelbead. In some cases, another species comprising a labile bond may belinked to a gel bead after gel bead formation via, for example, anactivated functional group of the gel bead as described above. As willbe appreciated, barcodes that are releasably, cleavably or reversiblyattached to the beads described herein include barcodes that arereleased or releasable through cleavage of a linkage between the barcodemolecule and the bead, or that are released through degradation of theunderlying bead itself, allowing the barcodes to be accessed oraccessible by other reagents, or both.

In addition to thermally cleavable bonds, disulfide bonds and UVsensitive bonds, other non-limiting examples of labile bonds that may becoupled to a precursor or bead include an ester linkage (e.g., cleavablewith an acid, a base, or hydroxylamine), a vicinal diol linkage (e.g.,cleavable via sodium periodate), a Diels-Alder linkage (e.g., cleavablevia heat), a sulfone linkage (e.g., cleavable via a base), a silyl etherlinkage (e.g., cleavable via an acid), a glycosidic linkage (e.g.,cleavable via an amylase), a peptide linkage (e.g., cleavable via aprotease), or a phosphodiester linkage (e.g., cleavable via a nuclease(e.g., DNAase)). A bond may be cleavable via other nucleic acid moleculetargeting enzymes, such as restriction enzymes (e.g., restrictionendonucleases), as described further below.

Species may be encapsulated in beads during bead generation (e.g.,during polymerization of precursors). Such species may or may notparticipate in polymerization. Such species may be entered intopolymerization reaction mixtures such that generated beads comprise thespecies upon bead formation. In some cases, such species may be added tothe gel beads after formation. Such species may include, for example,nucleic acid molecules (e.g., oligonucleotides), reagents for a nucleicacid amplification reaction (e.g., primers, polymerases, dNTPs,co-factors (e.g., ionic co-factors), buffers) including those describedherein, reagents for enzymatic reactions (e.g., enzymes, co-factors,substrates, buffers), reagents for nucleic acid modification reactionssuch as polymerization, ligation, or digestion, and/or reagents fortemplate preparation (e.g., tagmentation) for one or more sequencingplatforms (e.g., Nextera® for Illumina®). Such species may include oneor more enzymes described herein, including without limitation,polymerase, reverse transcriptase, restriction enzymes (e.g.,endonuclease), transposase, ligase, proteinase K, DNAse, etc. Suchspecies may include one or more reagents described elsewhere herein(e.g., lysis agents, inhibitors, inactivating agents, chelating agents,stimulus). Trapping of such species may be controlled by the polymernetwork density generated during polymerization of precursors, controlof ionic charge within the gel bead (e.g., via ionic species linked topolymerized species), or by the release of other species. Encapsulatedspecies may be released from a bead upon bead degradation and/or byapplication of a stimulus capable of releasing the species from thebead. Alternatively or in addition, species may be partitioned in apartition (e.g., droplet) during or subsequent to partition formation.Such species may include, without limitation, the abovementioned speciesthat may also be encapsulated in a bead.

A degradable bead may comprise one or more species with a labile bondsuch that, when the bead/species is exposed to the appropriate stimuli,the bond is broken and the bead degrades. The labile bond may be achemical bond (e.g., covalent bond, ionic bond) or may be another typeof physical interaction (e.g., van der Waals interactions, dipole-dipoleinteractions, etc.). In some cases, a crosslinker used to generate abead may comprise a labile bond. Upon exposure to the appropriateconditions, the labile bond can be broken and the bead degraded. Forexample, upon exposure of a polyacrylamide gel bead comprising cystaminecrosslinkers to a reducing agent, the disulfide bonds of the cystaminecan be broken and the bead degraded.

A degradable bead may be useful in more quickly releasing an attachedspecies (e.g., a nucleic acid molecule, a barcode sequence, a primer,etc) from the bead when the appropriate stimulus is applied to the beadas compared to a bead that does not degrade. For example, for a speciesbound to an inner surface of a porous bead or in the case of anencapsulated species, the species may have greater mobility andaccessibility to other species in solution upon degradation of the bead.In some cases, a species may also be attached to a degradable bead via adegradable linker (e.g., disulfide linker). The degradable linker mayrespond to the same stimuli as the degradable bead or the two degradablespecies may respond to different stimuli. For example, a barcodesequence may be attached, via a disulfide bond, to a polyacrylamide beadcomprising cystamine. Upon exposure of the barcoded-bead to a reducingagent, the bead degrades and the barcode sequence is released uponbreakage of both the disulfide linkage between the barcode sequence andthe bead and the disulfide linkages of the cystamine in the bead.

Where degradable beads are provided, it may be beneficial to avoidexposing such beads to the stimulus or stimuli that cause suchdegradation prior to a given time, in order to, for example, avoidpremature bead degradation and issues that arise from such degradation,including for example poor flow characteristics and aggregation. By wayof example, where beads comprise reducible cross-linking groups, such asdisulfide groups, it will be desirable to avoid contacting such beadswith reducing agents, e.g., DTT or other disulfide cleaving reagents. Insuch cases, treatment to the beads described herein will, in some casesbe provided free of reducing agents, such as DTT. Because reducingagents are often provided in commercial enzyme preparations, it may bedesirable to provide reducing agent free (or DTT free) enzymepreparations in treating the beads described herein. Examples of suchenzymes include, e.g., polymerase enzyme preparations, reversetranscriptase enzyme preparations, ligase enzyme preparations, as wellas many other enzyme preparations that may be used to treat the beadsdescribed herein. The terms “reducing agent free” or “DTT free”preparations can refer to a preparation having less than about ⅒th, lessthan about 1/50th, or even less than about 1/100th of the lower rangesfor such materials used in degrading the beads. For example, for DTT,the reducing agent free preparation can have less than about 0.01millimolar (mM), 0.005 mM, 0.001 mM DTT, 0.0005 mM DTT, or even lessthan about 0.0001 mM DTT. In many cases, the amount of DTT can beundetectable.

Numerous chemical triggers may be used to trigger the degradation ofbeads. Examples of these chemical changes may include, but are notlimited to pH-mediated changes to the integrity of a component withinthe bead, degradation of a component of a bead via cleavage ofcross-linked bonds, and depolymerization of a component of a bead.

In some embodiments, a bead may be formed from materials that comprisedegradable chemical crosslinkers, such as BAC or cystamine. Degradationof such degradable crosslinkers may be accomplished through a number ofmechanisms. In some examples, a bead may be contacted with a chemicaldegrading agent that may induce oxidation, reduction or other chemicalchanges. For example, a chemical degrading agent may be a reducingagent, such as dithiothreitol (DTT). Additional examples of reducingagents may include β-mercaptoethanol, (2S)-2-amino-1,4-dimercaptobutane(dithiobutylamine or DTBA), tris(2-carboxyethyl) phosphine (TCEP), orcombinations thereof. A reducing agent may degrade the disulfide bondsformed between gel precursors forming the bead, and thus, degrade thebead. In other cases, a change in pH of a solution, such as an increasein pH, may trigger degradation of a bead. In other cases, exposure to anaqueous solution, such as water, may trigger hydrolytic degradation, andthus degradation of the bead. In some cases, any combination of stimulimay trigger degradation of a bead. For example, a change in pH mayenable a chemical agent (e.g., DTT) to become an effective reducingagent.

Beads may also be induced to release their contents upon the applicationof a thermal stimulus. A change in temperature can cause a variety ofchanges to a bead. For example, heat can cause a solid bead to liquefy.A change in heat may cause melting of a bead such that a portion of thebead degrades. In other cases, heat may increase the internal pressureof the bead components such that the bead ruptures or explodes. Heat mayalso act upon heat-sensitive polymers used as materials to constructbeads.

Any suitable agent may degrade beads. In some embodiments, changes intemperature or pH may be used to degrade thermo-sensitive orpH-sensitive bonds within beads. In some embodiments, chemical degradingagents may be used to degrade chemical bonds within beads by oxidation,reduction or other chemical changes. For example, a chemical degradingagent may be a reducing agent, such as DTT, wherein DTT may degrade thedisulfide bonds formed between a crosslinker and gel precursors, thusdegrading the bead. In some embodiments, a reducing agent may be addedto degrade the bead, which may or may not cause the bead to release itscontents. Examples of reducing agents may include dithiothreitol (DTT),β-mercaptoethanol, (2S)-2-amino-1,4-dimercaptobutane (dithiobutylamineor DTBA), tris(2-carboxyethyl) phosphine (TCEP), or combinationsthereof. The reducing agent may be present at a concentration of about0.1 mM, 0.5 mM, 1 mM, 5 mM, 10 mM. The reducing agent may be present ata concentration of at least about 0.1 mM, 0.5 mM, 1 mM, 5 mM, 10 mM, orgreater than 10 mM. The reducing agent may be present at concentrationof at most about 10 mM, 5 mM, 1 mM, 0.5 mM, 0.1 mM, or less.

Although FIG. 1 and FIG. 2 have been described in terms of providingsubstantially singly occupied partitions, above, in certain cases, itmay be desirable to provide multiply occupied partitions, e.g.,containing two, three, four or more cells and/or microcapsules (e.g.,beads) comprising barcoded nucleic acid molecules (e.g.,oligonucleotides) within a single partition. Accordingly, as notedabove, the flow characteristics of the biological particle and/or beadcontaining fluids and partitioning fluids may be controlled to providefor such multiply occupied partitions. In particular, the flowparameters may be controlled to provide a given occupancy rate atgreater than about 50% of the partitions, greater than about 75%, and insome cases greater than about 80%, 90%, 95%, or higher.

In some cases, additional microcapsules can be used to deliveradditional reagents to a partition. In such cases, it may beadvantageous to introduce different beads into a common channel ordroplet generation junction, from different bead sources (e.g.,containing different associated reagents) through different channelinlets into such common channel or droplet generation junction (e.g.,junction 210). In such cases, the flow and frequency of the differentbeads into the channel or junction may be controlled to provide for acertain ratio of microcapsules from each source, while ensuring a givenpairing or combination of such beads into a partition with a givennumber of biological particles (e.g., one biological particle and onebead per partition).

The partitions described herein may comprise small volumes, for example,less than about 10 microliters (µL), 5 µL, 1 µL, 500 nanoliters (nL),100 nL, 50 nL, 900 picoliters (pL), 800 pL, 700 pL, 600 pL, 500 pL, 400pL, 300 pL, 200 pL, 100 pL, 50 pL, 20 pL, 10 pL, 1 pL, or less.

For example, in the case of droplet based partitions, the droplets mayhave overall volumes that are less than about 1000 pL, 900 pL, 800 pL,700 pL, 600 pL, 500 pL, 400 pL, 300 pL, 200 pL, 100 pL, 50 pL, 20 pL, 10pL, 1 pL, or less. Where co-partitioned with microcapsules, it will beappreciated that the sample fluid volume, e.g., including co-partitionedbiological particles and/or beads, within the partitions may be lessthan about 90% of the above described volumes, less than about 80%, lessthan about 70%, less than about 60%, less than about 50%, less thanabout 40%, less than about 30%, less than about 20%, or less than about10% of the above described volumes.

As is described elsewhere herein, partitioning species may generate apopulation or plurality of partitions. In such cases, any suitablenumber of partitions can be generated or otherwise provided. Forexample, at least about 1,000 partitions, at least about 5,000partitions, at least about 10,000 partitions, at least about 50,000partitions, at least about 100,000 partitions, at least about 500,000partitions, at least about 1,000 ,000 partitions, at least about 5,000,000 partitions at least about 10,000 ,000 partitions, at least about50,000 ,000 partitions, at least about 100,000 ,000 partitions, at leastabout 500,000 ,000 partitions, at least about 1,000 ,000,000 partitions,or more partitions can be generated or otherwise provided. Moreover, theplurality of partitions may comprise both unoccupied partitions (e.g.,empty partitions) and occupied partitions.

Reagents

In accordance with certain aspects, biological particles may bepartitioned along with lysis reagents in order to release the contentsof the biological particles within the partition. In such cases, thelysis agents can be contacted with the biological particle suspensionconcurrently with, or immediately prior to, the introduction of thebiological particles into the partitioning junction/droplet generationzone (e.g., junction 210), such as through an additional channel orchannels upstream of the channel junction. In accordance with otheraspects, additionally or alternatively, biological particles may bepartitioned along with other reagents, as will be described furtherbelow.

FIG. 3 shows an example of a microfluidic channel structure 300 forco-partitioning biological particles and reagents. The channel structure300 can include channel segments 301, 302, 304, 306 and 308. Channelsegments 301 and 302 communicate at a first channel junction 309.Channel segments 302, 304, 306, and 308 communicate at a second channeljunction 310.

In an example operation, the channel segment 301 may transport anaqueous fluid 312 that includes a plurality of biological particles 314along the channel segment 301 into the second junction 310. As analternative or in addition to, channel segment 301 may transport beads(e.g., gel beads). The beads may comprise barcode molecules.

For example, the channel segment 301 may be connected to a reservoircomprising an aqueous suspension of biological particles 314. Upstreamof, and immediately prior to reaching, the second junction 310, thechannel segment 301 may meet the channel segment 302 at the firstjunction 309. The channel segment 302 may transport a plurality ofreagents 315 (e.g., lysis agents) suspended in the aqueous fluid 312along the channel segment 302 into the first junction 309. For example,the channel segment 302 may be connected to a reservoir comprising thereagents 315. After the first junction 309, the aqueous fluid 312 in thechannel segment 301 can carry both the biological particles 314 and thereagents 315 towards the second junction 310. In some instances, theaqueous fluid 312 in the channel segment 301 can include one or morereagents, which can be the same or different reagents as the reagents315. A second fluid 316 that is immiscible with the aqueous fluid 312(e.g., oil) can be delivered to the second junction 310 from each ofchannel segments 304 and 306. Upon meeting of the aqueous fluid 312 fromthe channel segment 301 and the second fluid 316 from each of channelsegments 304 and 306 at the second channel junction 310, the aqueousfluid 312 can be partitioned as discrete droplets 318 in the secondfluid 316 and flow away from the second junction 310 along channelsegment 308. The channel segment 308 may deliver the discrete droplets318 to an outlet reservoir fluidly coupled to the channel segment 308,where they may be harvested.

The second fluid 316 can comprise an oil, such as a fluorinated oil,that includes a fluorosurfactant for stabilizing the resulting droplets,for example, inhibiting subsequent coalescence of the resulting droplets318.

A discrete droplet generated may include an individual biologicalparticle 314 and/or one or more reagents 315. In some instances, adiscrete droplet generated may include a barcode carrying bead (notshown), such as via other microfluidics structures described elsewhereherein. In some instances, a discrete droplet may be unoccupied (e.g.,no reagents, no biological particles).

Beneficially, when lysis reagents and biological particles areco-partitioned, the lysis reagents can facilitate the release of thecontents of the biological particles within the partition. The contentsreleased in a partition may remain discrete from the contents of otherpartitions.

As will be appreciated, the channel segments described herein may becoupled to any of a variety of different fluid sources or receivingcomponents, including reservoirs, tubing, manifolds, or fluidiccomponents of other systems. As will be appreciated, the microfluidicchannel structure 300 may have other geometries. For example, amicrofluidic channel structure can have more than two channel junctions.For example, a microfluidic channel structure can have 2, 3, 4, 5channel segments or more each carrying the same or different types ofbeads, reagents, and/or biological particles that meet at a channeljunction. Fluid flow in each channel segment may be controlled tocontrol the partitioning of the different elements into droplets. Fluidmay be directed flow along one or more channels or reservoirs via one ormore fluid flow units. A fluid flow unit can comprise compressors (e.g.,providing positive pressure), pumps (e.g., providing negative pressure),actuators, and the like to control flow of the fluid. Fluid may also orotherwise be controlled via applied pressure differentials, centrifugalforce, magnetic force, electrokinetic pumping, vacuum, capillary orgravity flow, or the like.

Examples of lysis agents include bioactive reagents, such as lysisenzymes that are used for lysis of different cell types, e.g.,gram-positive or gram-negative bacteria, plants, yeast, mammalian, etc.,such as lysozymes, achromopeptidase, lysostaphin, labiase, kitalase,lyticase, and a variety of other lysis enzymes available from, e.g.,Sigma-Aldrich, Inc. (St Louis, MO), as well as other commerciallyavailable lysis enzymes. Other lysis agents may additionally oralternatively be co-partitioned with the biological particles to causethe release of the biological particle’s contents into the partitions.For example, in some cases, surfactant-based lysis solutions may be usedto lyse cells, although these may be less desirable for emulsion-basedsystems where the surfactants can interfere with stable emulsions. Insome cases, lysis solutions may include non-ionic surfactants such as,for example, TritonX-100, CHAPS, and Tween 20. In some cases, lysissolutions may include ionic surfactants such as, for example, sarcosyland sodium dodecyl sulfate (SDS). In some cases, lysis may be achievedthrough osmotic pressure, e.g., using a hypotonic lysis buffer.Electroporation, thermal, acoustic or mechanical cellular disruption mayalso be used in certain cases, e.g., non-emulsion based partitioningsuch as encapsulation of biological particles that may be in addition toor in place of droplet partitioning, where any pore size of theencapsulate is sufficiently small to retain nucleic acid fragments of agiven size, following cellular disruption.

Alternatively or in addition to the lysis agents co-partitioned with thebiological particles described above, other reagents can also beco-partitioned with the biological particles, including, for example,DNase and RNase, inactivating agents or inhibitors, such as proteinase Kand/or other protease inhibitors, phosphatase inhibitors, chelatingagents, such as EDTA, and other reagents employed in removing orotherwise reducing negative activity or impact of different cell lysatecomponents on subsequent processing of nucleic acids. In addition, inthe case of encapsulated biological particles, the biological particlesmay be exposed to an appropriate stimulus to release the biologicalparticles or their contents from a co-partitioned microcapsule. Forexample, in some cases, a chemical stimulus may be co-partitioned alongwith an encapsulated biological particle to allow for the degradation ofthe microcapsule and release of the cell or its contents into the largerpartition. In some cases, this stimulus may be the same as the stimulusdescribed elsewhere herein for release of nucleic acid molecules (e.g.,oligonucleotides) from their respective microcapsule (e.g., bead). Inalternative aspects, this may be a different and non-overlappingstimulus, in order to allow an encapsulated biological particle to bereleased into a partition at a different time from the release ofnucleic acid molecules into the same partition.

Additional reagents may also be co-partitioned with the biologicalparticles, such as endonucleases to fragment a biological particle’sDNA, DNA polymerase enzymes and dNTPs used to amplify the biologicalparticle’s nucleic acid fragments and to attach the barcode moleculartags to the amplified fragments. Other enzymes may be co-partitioned,including without limitation, polymerase, transposase, ligase,proteinase K, DNAse, etc. Additional reagents may also include reversetranscriptase enzymes, including enzymes with terminal transferaseactivity, primers and oligonucleotides, and switch oligonucleotides(also referred to herein as “switch oligos” or “template switchingoligonucleotides”) which can be used for template switching. In somecases, template switching can be used to increase the length of a cDNA.In some cases, template switching can be used to append a predefinednucleic acid sequence to the cDNA. In an example of template switching,cDNA can be generated from reverse transcription of a template, e.g.,cellular mRNA, where a reverse transcriptase with terminal transferaseactivity can add additional nucleotides, e.g., polyC, to the cDNA in atemplate independent manner. Switch oligos can include sequencescomplementary to the additional nucleotides, e.g., polyG. The additionalnucleotides (e.g., polyC) on the cDNA can hybridize to the additionalnucleotides (e.g., polyG) on the switch oligo, whereby the switch oligocan be used by the reverse transcriptase as template to further extendthe cDNA. Template switching oligonucleotides may comprise ahybridization region and a template region. The hybridization region cancomprise any sequence capable of hybridizing to the target. In somecases, as previously described, the hybridization region comprises aseries of G bases to complement the overhanging C bases at the 3′ end ofa cDNA molecule. The series of G bases may comprise 1 G base, 2 G bases,3 G bases, 4 G bases, 5 G bases or more than5 G bases. The templatesequence can comprise any sequence to be incorporated into the cDNA. Insome cases, the template region comprises at least 1 (e.g., at least 2,3, 4, 5 or more) tag sequences and/or functional sequences. Switcholigos may comprise deoxyribonucleic acids; ribonucleic acids; modifiednucleic acids including 2-Aminopurine, 2,6-Diaminopurine (2-Amino-dA),inverted dT, 5-Methyl dC, 2′-deoxyInosine, Super T(5-hydroxybutynl-2′-deoxyuridine), Super G (8-aza-7-deazaguanosine),locked nucleic acids (LNAs), unlocked nucleic acids (UNAs, e.g., UNA-A,UNA-U, UNA-C, UNA-G), Iso-dG, Iso-dC, 2′ Fluoro bases (e.g., Fluoro C,Fluoro U, Fluoro A, and Fluoro G), or any combination.

In some cases, the length of a switch oligo may be at least about 2, 3,4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22,23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40,41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58,59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76,77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94,95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109,110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123,124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137,138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151,152, 153, 154, 155, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165,166, 167, 168, 169, 170, 171, 172, 173, 174, 175, 176, 177, 178, 179,180, 181, 182, 183, 184, 185, 186, 187, 188, 189, 190, 191, 192, 193,194, 195, 196, 197 , 198, 199, 200, 201, 202, 203, 204, 205, 206, 207,208, 209, 210, 211, 212, 213, 214, 215, 216, 217, 218, 219, 220, 221,222, 223, 224, 225, 226, 227, 228, 229, 230, 231, 232, 233, 234, 235,236, 237, 238, 239, 240, 241, 242, 243, 244, 245, 246, 247, 248, 249 or250 nucleotides or longer.

In some cases, the length of a switch oligo may be at most about 2, 3,4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22,23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40,41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58,59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76,77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94,95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109,110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123,124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137,138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151,152, 153, 154, 155, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165,166, 167, 168, 169, 170, 171, 172, 173, 174, 175, 176, 177, 178, 179,180, 181, 182, 183, 184, 185, 186, 187, 188, 189, 190, 191, 192, 193,194, 195, 196, 197 , 198, 199, 200, 201, 202, 203, 204, 205, 206, 207,208, 209, 210, 211, 212, 213, 214, 215, 216, 217, 218, 219, 220, 221,222, 223, 224, 225, 226, 227, 228, 229, 230, 231, 232, 233, 234, 235,236, 237, 238, 239, 240, 241, 242, 243, 244, 245, 246, 247, 248, 249 or250 nucleotides.

Once the contents of the cells are released into their respectivepartitions, the macromolecular components (e.g., macromolecularconstituents of biological particles, such as RNA, DNA, or proteins)contained therein may be further processed within the partitions. Inaccordance with the methods and systems described herein, themacromolecular component contents of individual biological particles canbe provided with unique identifiers such that, upon characterization ofthose macromolecular components they may be attributed as having beenderived from the same biological particle or particles. The ability toattribute characteristics to individual biological particles or groupsof biological particles is provided by the assignment of uniqueidentifiers specifically to an individual biological particle or groupsof biological particles. Unique identifiers, e.g., in the form ofnucleic acid barcodes can be assigned or associated with individualbiological particles or populations of biological particles, in order totag or label the biological particle’s macromolecular components (and asa result, its characteristics) with the unique identifiers. These uniqueidentifiers can then be used to attribute the biological particle’scomponents and characteristics to an individual biological particle orgroup of biological particles.

In some aspects, this is performed by co-partitioning the individualbiological particle or groups of biological particles with the uniqueidentifiers, such as described above (with reference to FIG. 2 ). Insome aspects, the unique identifiers are provided in the form of nucleicacid molecules (e.g., oligonucleotides) that comprise nucleic acidbarcode sequences that may be attached to or otherwise associated withthe nucleic acid contents of individual biological particle, or to othercomponents of the biological particle, and particularly to fragments ofthose nucleic acids. The nucleic acid molecules are partitioned suchthat as between nucleic acid molecules in a given partition, the nucleicacid barcode sequences contained therein are the same, but as betweendifferent partitions, the nucleic acid molecule can, and do havediffering barcode sequences, or at least represent a large number ofdifferent barcode sequences across all of the partitions in a givenanalysis. In some aspects, only one nucleic acid barcode sequence can beassociated with a given partition, although in some cases, two or moredifferent barcode sequences may be present.

The nucleic acid barcode sequences can include from about 6 to about 20or more nucleotides within the sequence of the nucleic acid molecules(e.g., oligonucleotides). The nucleic acid barcode sequences can includefrom about 6 to about 20, 30, 40, 50, 60, 70, 80, 90, 100 or morenucleotides. In some cases, the length of a barcode sequence may beabout 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 nucleotidesor longer. In some cases, the length of a barcode sequence may be atleast about 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20nucleotides or longer. In some cases, the length of a barcode sequencemay be at most about 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19,20 nucleotides or shorter. These nucleotides may be completelycontiguous, i.e., in a single stretch of adjacent nucleotides, or theymay be separated into two or more separate subsequences that areseparated by 1 or more nucleotides. In some cases, separated barcodesubsequences can be from about 4 to about 16 nucleotides in length. Insome cases, the barcode subsequence may be about 4, 5, 6, 7, 8, 9, 10,11, 12, 13, 14, 15, 16 nucleotides or longer. In some cases, the barcodesubsequence may be at least about 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14,15, 16 nucleotides or longer. In some cases, the barcode subsequence maybe at most about 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16nucleotides or shorter.

The co-partitioned nucleic acid molecules can also comprise otherfunctional sequences useful in the processing of the nucleic acids fromthe co-partitioned biological particles. These sequences include, e.g.,targeted or random/universal amplification primer sequences foramplifying the genomic DNA from the individual biological particleswithin the partitions while attaching the associated barcode sequences,sequencing primers or primer recognition sites, hybridization or probingsequences, e.g., for identification of presence of the sequences or forpulling down and/or collecting barcoded nucleic acids, or any of anumber of other potential functional sequences. Other mechanisms ofco-partitioning oligonucleotides may also be employed, including, e.g.,coalescence of two or more droplets, where one droplet containsoligonucleotides, or microdispensing of oligonucleotides intopartitions, e.g., droplets within microfluidic systems.

In an example, microcapsules, such as beads, are provided that eachinclude large numbers of the above described barcoded nucleic acidmolecules (e.g., barcoded oligonucleotides) releasably attached to thebeads, where all of the nucleic acid molecules attached to a particularbead will include the same nucleic acid barcode sequence, but where alarge number of diverse barcode sequences are represented across thepopulation of beads used. In some embodiments, hydrogel beads, e.g.,comprising polyacrylamide polymer matrices, are used as a solid supportand delivery vehicle for the nucleic acid molecules into the partitions,as they are capable of carrying large numbers of nucleic acid molecules,and may be configured to release those nucleic acid molecules uponexposure to a particular stimulus, as described elsewhere herein. Insome cases, the population of beads provides a diverse barcode sequencelibrary that includes at least about 1,000 different barcode sequences,at least about 5,000 different barcode sequences, at least about 10,000different barcode sequences, at least about 50,000 different barcodesequences, at least about 100,000 different barcode sequences, at leastabout 1,000 ,000 different barcode sequences, at least about 5,000 ,000different barcode sequences, or at least about 10,000 ,000 differentbarcode sequences, or more. Additionally, each bead can be provided withlarge numbers of nucleic acid (e.g., oligonucleotide) moleculesattached. In particular, the number of molecules of nucleic acidmolecules including the barcode sequence on an individual bead can be atleast about 1,000 nucleic acid molecules, at least about5,000 nucleicacid molecules, at least about 10,000 nucleic acid molecules, at leastabout 50,000 nucleic acid molecules, at least about 100,000 nucleic acidmolecules, at least about 500,000 nucleic acids, at least about 1,000,000 nucleic acid molecules, at least about 5,000 ,000 nucleic acidmolecules, at least about 10,000 ,000 nucleic acid molecules, at leastabout 50,000 ,000 nucleic acid molecules, at least about 100,000 ,000nucleic acid molecules, at least about 250,000 ,000 nucleic acidmolecules and in some cases at least about 1 billion nucleic acidmolecules, or more. Nucleic acid molecules of a given bead can includeidentical (or common) barcode sequences, different barcode sequences, ora combination of both. Nucleic acid molecules of a given bead caninclude multiple sets of nucleic acid molecules. Nucleic acid moleculesof a given set can include identical barcode sequences. The identicalbarcode sequences can be different from barcode sequences of nucleicacid molecules of another set.

Moreover, when the population of beads is partitioned, the resultingpopulation of partitions can also include a diverse barcode library thatincludes at least about 1,000 different barcode sequences, at leastabout 5,000 different barcode sequences, at least about 10,000 differentbarcode sequences, at least at least about 50,000 different barcodesequences, at least about 100,000 different barcode sequences, at leastabout 1,000 ,000 different barcode sequences, at least about 5,000 ,000different barcode sequences, or at least about 10,000 ,000 differentbarcode sequences. Additionally, each partition of the population caninclude at least about 1,000 nucleic acid molecules, at least about5,000 nucleic acid molecules, at least about 10,000 nucleic acidmolecules, at least about 50,000 nucleic acid molecules, at least about100,000 nucleic acid molecules, at least about 500,000 nucleic acids, atleast about 1,000 ,000 nucleic acid molecules, at least about 5,000 ,000nucleic acid molecules, at least about 10,000 ,000 nucleic acidmolecules, at least about 50,000 ,000 nucleic acid molecules, at leastabout 100,000 ,000 nucleic acid molecules, at least about 250,000 ,000nucleic acid molecules and in some cases at least about 1 billionnucleic acid molecules.

In some cases, it may be desirable to incorporate multiple differentbarcodes within a given partition, either attached to a single ormultiple beads within the partition. For example, in some cases, amixed, but known set of barcode sequences may provide greater assuranceof identification in the subsequent processing, e.g., by providing astronger address or attribution of the barcodes to a given partition, asa duplicate or independent confirmation of the output from a givenpartition.

The nucleic acid molecules (e.g., oligonucleotides) are releasable fromthe beads upon the application of a particular stimulus to the beads. Insome cases, the stimulus may be a photo-stimulus, e.g., through cleavageof a photo-labile linkage that releases the nucleic acid molecules. Inother cases, a thermal stimulus may be used, where elevation of thetemperature of the beads environment will result in cleavage of alinkage or other release of the nucleic acid molecules from the beads.In still other cases, a chemical stimulus can be used that cleaves alinkage of the nucleic acid molecules to the beads, or otherwise resultsin release of the nucleic acid molecules from the beads. In one case,such compositions include the polyacrylamide matrices described abovefor encapsulation of biological particles, and may be degraded forrelease of the attached nucleic acid molecules through exposure to areducing agent, such as DTT.

In some aspects, provided are systems and methods for controlledpartitioning. Droplet size may be controlled by adjusting certaingeometric features in channel architecture (e.g., microfluidics channelarchitecture). For example, an expansion angle, width, and/or length ofa channel may be adjusted to control droplet size.

FIG. 4 shows an example of a microfluidic channel structure for thecontrolled partitioning of beads into discrete droplets. A channelstructure 400 can include a channel segment 402 communicating at achannel junction 406 (or intersection) with a reservoir 404. Thereservoir 404 can be a chamber. Any reference to “reservoir,” as usedherein, can also refer to a “chamber.” In operation, an aqueous fluid408 that includes suspended beads 412 may be transported along thechannel segment 402 into the junction 406 to meet a second fluid 410that is immiscible with the aqueous fluid 408 in the reservoir 404 tocreate droplets 416, 418 of the aqueous fluid 408 flowing into thereservoir 404. At the junction 406 where the aqueous fluid 408 and thesecond fluid 410 meet, droplets can form based on factors such as thehydrodynamic forces at the junction 406, flow rates of the two fluids408, 410, fluid properties, and certain geometric parameters (e.g., w,h₀, α, etc.) of the channel structure 400. A plurality of droplets canbe collected in the reservoir 404 by continuously injecting the aqueousfluid 408 from the channel segment 402 through the junction 406.

A discrete droplet generated may include a bead (e.g., as in occupieddroplets 416). Alternatively, a discrete droplet generated may includemore than one bead. Alternatively, a discrete droplet generated may notinclude any beads (e.g., as in unoccupied droplet 418). In someinstances, a discrete droplet generated may contain one or morebiological particles, as described elsewhere herein. In some instances,a discrete droplet generated may comprise one or more reagents, asdescribed elsewhere herein.

In some instances, the aqueous fluid 408 can have a substantiallyuniform concentration or frequency of beads 412. The beads 412 can beintroduced into the channel segment 402 from a separate channel (notshown in FIG. 4 ). The frequency of beads 412 in the channel segment 402may be controlled by controlling the frequency in which the beads 412are introduced into the channel segment 402 and/or the relative flowrates of the fluids in the channel segment 402 and the separate channel.In some instances, the beads can be introduced into the channel segment402 from a plurality of different channels, and the frequency controlledaccordingly.

In some instances, the aqueous fluid 408 in the channel segment 402 cancomprise biological particles (e.g., described with reference to FIGS. 1and 2 ). In some instances, the aqueous fluid 408 can have asubstantially uniform concentration or frequency of biologicalparticles. As with the beads, the biological particles can be introducedinto the channel segment 402 from a separate channel. The frequency orconcentration of the biological particles in the aqueous fluid 408 inthe channel segment 402 may be controlled by controlling the frequencyin which the biological particles are introduced into the channelsegment 402 and/or the relative flow rates of the fluids in the channelsegment 402 and the separate channel. In some instances, the biologicalparticles can be introduced into the channel segment 402 from aplurality of different channels, and the frequency controlledaccordingly. In some instances, a first separate channel can introducebeads and a second separate channel can introduce biological particlesinto the channel segment 402. The first separate channel introducing thebeads may be upstream or downstream of the second separate channelintroducing the biological particles.

The second fluid 410 can comprise an oil, such as a fluorinated oil,that includes a fluorosurfactant for stabilizing the resulting droplets,for example, inhibiting subsequent coalescence of the resultingdroplets.

In some instances, the second fluid 410 may not be subjected to and/ordirected to any flow in or out of the reservoir 404. For example, thesecond fluid 410 may be substantially stationary in the reservoir 404.In some instances, the second fluid 410 may be subjected to flow withinthe reservoir 404, but not in or out of the reservoir 404, such as viaapplication of pressure to the reservoir 404 and/or as affected by theincoming flow of the aqueous fluid 408 at the junction 406.Alternatively, the second fluid 410 may be subjected and/or directed toflow in or out of the reservoir 404. For example, the reservoir 404 canbe a channel directing the second fluid 410 from upstream to downstream,transporting the generated droplets.

The channel structure 400 at or near the junction 406 may have certaingeometric features that at least partly determine the sizes of thedroplets formed by the channel structure 400. The channel segment 402can have a height, h₀ and width, w, at or near the junction 406. By wayof example, the channel segment 402 can comprise a rectangularcross-section that leads to a reservoir 404 having a wider cross-section(such as in width or diameter). Alternatively, the cross-section of thechannel segment 402 can be other shapes, such as a circular shape,trapezoidal shape, polygonal shape, or any other shapes. The top andbottom walls of the reservoir 404 at or near the junction 406 can beinclined at an expansion angle, α. The expansion angle, α, allows thetongue (portion of the aqueous fluid 408 leaving channel segment 402 atjunction 406 and entering the reservoir 404 before droplet formation) toincrease in depth and facilitate decrease in curvature of theintermediately formed droplet. Droplet size may decrease with increasingexpansion angle. The resulting droplet radius, R_(d), may be predictedby the following equation for the aforementioned geometric parameters ofh₀, w, and α:

$R_{d} \approx 0.44( {1 + 2.2\sqrt{\tan\alpha}\frac{w}{h_{0}}} )\frac{h_{0}}{\sqrt{\tan\alpha}}$

By way of example, for a channel structure with w = 21 µm, h = 21 µm,and α = 3°, the predicted droplet diameter is 121 µm. In anotherexample, for a channel structure with w = 25 µm, h = 25 µm, and α = 5°,the predicted droplet diameter is 123 µm. In another example, for achannel structure with w = 28 µm, h = 28 µm, and α = 7°, the predicteddroplet diameter is 124 µm.

In some instances, the expansion angle, α, may be between a range offrom about 0.5° to about 4°, from about 0.1° to about 10°, or from about0° to about 90°. For example, the expansion angle can be at least about0.01°, 0.1°, 0.2°, 0.3°, 0.4°, 0.5°, 0.6°, 0.7°, 0.8°, 0.9°, 1°, 2°, 3°,4°, 5°, 6°, 7°, 8°, 9°, 10°, 15°, 20°, 25°, 30°, 35°, 40°, 45°, 50°,55°, 60°, 65°, 70°, 75°, 80°, 85°, or higher. In some instances, theexpansion angle can be at most about 89°, 88°, 87°, 86°, 85°, 84°, 83°,82°, 81°, 80°, 75°, 70°, 65°, 60°, 55°, 50°, 45°, 40°, 35° 30°, 25°,20°, 15° 10°, 9° 8° 7° 6°, 5°, 4°, 3°, 2°, 1°, 0.1°, 0.01°, or less. Insome instances, the width, w, can be between a range of from about 100micrometers (µm) to about 500 µm. In some instances, the width, w, canbe between a range of from about 10 µm to about 200 µm. Alternatively,the width can be less than about 10 µm. Alternatively, the width can begreater than about 500 µm. In some instances, the flow rate of theaqueous fluid 408 entering the junction 406 can be between about 0.04microliters (µL)/minute (min) and about 40 µL/min. In some instances,the flow rate of the aqueous fluid 408 entering the junction 406 can bebetween about 0.01 microliters (µL)/minute (min) and about 100 µL/min.Alternatively, the flow rate of the aqueous fluid 408 entering thejunction 406 can be less than about 0.01 µL/min. Alternatively, the flowrate of the aqueous fluid 408 entering the junction 406 can be greaterthan about 40 µL/min, such as 45 µL/min, 50 µL/min, 55 µL/min, 60µL/min, 65 µL/min, 70 µL/min, 75 µL/min, 80 µL/min, 85 µL/min, 90µL/min, 95 µL/min, 100 µL/min, 110 µL/min, 120 µL/min , 130 µL/min , 140µL/min, 150 µL/min, or greater. At lower flow rates, such as flow ratesof about less than or equal to 10 microliters/minute, the droplet radiusmay not be dependent on the flow rate of the aqueous fluid 408 enteringthe junction 406.

In some instances, at least about 50% of the droplets generated can haveuniform size. In some instances, at least about 55%, 60%, 65%, 70%, 75%,80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or greater of the dropletsgenerated can have uniform size. Alternatively, less than about 50% ofthe droplets generated can have uniform size.

The throughput of droplet generation can be increased by increasing thepoints of generation, such as increasing the number of junctions (e.g.,junction 406) between aqueous fluid 408 channel segments (e.g., channelsegment 402) and the reservoir 404. Alternatively or in addition, thethroughput of droplet generation can be increased by increasing the flowrate of the aqueous fluid 408 in the channel segment 402.

FIG. 5 shows an example of a microfluidic channel structure forincreased droplet generation throughput. A microfluidic channelstructure 500 can comprise a plurality of channel segments 502 and areservoir 504. Each of the plurality of channel segments 502 may be influid communication with the reservoir 504. The channel structure 500can comprise a plurality of channel junctions 506 between the pluralityof channel segments 502 and the reservoir 504. Each channel junction canbe a point of droplet generation. The channel segment 402 from thechannel structure 400 in FIG. 4 and any description to the componentsthereof may correspond to a given channel segment of the plurality ofchannel segments 502 in channel structure 500 and any description to thecorresponding components thereof. The reservoir 404 from the channelstructure 400 and any description to the components thereof maycorrespond to the reservoir 504 from the channel structure 500 and anydescription to the corresponding components thereof.

Each channel segment of the plurality of channel segments 502 maycomprise an aqueous fluid 508 that includes suspended beads 512. Thereservoir 504 may comprise a second fluid 510 that is immiscible withthe aqueous fluid 508. In some instances, the second fluid 510 may notbe subjected to and/or directed to any flow in or out of the reservoir504. For example, the second fluid 510 may be substantially stationaryin the reservoir 504. In some instances, the second fluid 510 may besubjected to flow within the reservoir 504, but not in or out of thereservoir 504, such as via application of pressure to the reservoir 504and/or as affected by the incoming flow of the aqueous fluid 508 at thejunctions. Alternatively, the second fluid 510 may be subjected and/ordirected to flow in or out of the reservoir 504. For example, thereservoir 504 can be a channel directing the second fluid 510 fromupstream to downstream, transporting the generated droplets.

In operation, the aqueous fluid 508 that includes suspended beads 512may be transported along the plurality of channel segments 502 into theplurality of junctions 506 to meet the second fluid 510 in the reservoir504 to create droplets 516, 518. A droplet may form from each channelsegment at each corresponding junction with the reservoir 504. At thejunction where the aqueous fluid 508 and the second fluid 510 meet,droplets can form based on factors such as the hydrodynamic forces atthe junction, flow rates of the two fluids 508, 510, fluid properties,and certain geometric parameters (e.g., w, h₀, α, etc.) of the channelstructure 500, as described elsewhere herein. A plurality of dropletscan be collected in the reservoir 504 by continuously injecting theaqueous fluid 508 from the plurality of channel segments 502 through theplurality of junctions 506. Throughput may significantly increase withthe parallel channel configuration of channel structure 500. Forexample, a channel structure having five inlet channel segmentscomprising the aqueous fluid 508 may generate droplets five times asfrequently than a channel structure having one inlet channel segment,provided that the fluid flow rate in the channel segments aresubstantially the same. The fluid flow rate in the different inletchannel segments may or may not be substantially the same. A channelstructure may have as many parallel channel segments as is practical andallowed for the size of the reservoir. For example, the channelstructure may have at least about 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30,40, 50, 60, 70, 80, 90, 100, 150, 500, 250, 300, 350, 400, 450, 500,600, 700, 800, 900, 1000, 1500, 5000 or more parallel or substantiallyparallel channel segments.

The geometric parameters, w, h₀, and α, may or may not be uniform foreach of the channel segments in the plurality of channel segments 502.For example, each channel segment may have the same or different widthsat or near its respective channel junction with the reservoir 504. Forexample, each channel segment may have the same or different height ator near its respective channel junction with the reservoir 504. Inanother example, the reservoir 504 may have the same or differentexpansion angle at the different channel junctions with the plurality ofchannel segments 502. When the geometric parameters are uniform,beneficially, droplet size may also be controlled to be uniform evenwith the increased throughput. In some instances, when it is desirableto have a different distribution of droplet sizes, the geometricparameters for the plurality of channel segments 502 may be variedaccordingly.

In some instances, at least about 50% of the droplets generated can haveuniform size. In some instances, at least about 55%, 60%, 65%, 70%, 75%,80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or greater of the dropletsgenerated can have uniform size. Alternatively, less than about 50% ofthe droplets generated can have uniform size.

FIG. 6 shows another example of a microfluidic channel structure forincreased droplet generation throughput. A microfluidic channelstructure 600 can comprise a plurality of channel segments 602 arrangedgenerally circularly around the perimeter of a reservoir 604. Each ofthe plurality of channel segments 602 may be in fluid communication withthe reservoir 604. The channel structure 600 can comprise a plurality ofchannel junctions 606 between the plurality of channel segments 602 andthe reservoir 604. Each channel junction can be a point of dropletgeneration. The channel segment 402 from the channel structure 400 inFIG. 4 and any description to the components thereof may correspond to agiven channel segment of the plurality of channel segments 602 inchannel structure 600 and any description to the correspondingcomponents thereof. The reservoir 404 from the channel structure 400 andany description to the components thereof may correspond to thereservoir 604 from the channel structure 600 and any description to thecorresponding components thereof.

Each channel segment of the plurality of channel segments 602 maycomprise an aqueous fluid 608 that includes suspended beads 612. Thereservoir 604 may comprise a second fluid 610 that is immiscible withthe aqueous fluid 608. In some instances, the second fluid 610 may notbe subjected to and/or directed to any flow in or out of the reservoir604. For example, the second fluid 610 may be substantially stationaryin the reservoir 604. In some instances, the second fluid 610 may besubjected to flow within the reservoir 604, but not in or out of thereservoir 604, such as via application of pressure to the reservoir 604and/or as affected by the incoming flow of the aqueous fluid 608 at thejunctions. Alternatively, the second fluid 610 may be subjected and/ordirected to flow in or out of the reservoir 604. For example, thereservoir 604 can be a channel directing the second fluid 610 fromupstream to downstream, transporting the generated droplets.

In operation, the aqueous fluid 608 that includes suspended beads 612may be transported along the plurality of channel segments 602 into theplurality of junctions 606 to meet the second fluid 610 in the reservoir604 to create a plurality of droplets 616. A droplet may form from eachchannel segment at each corresponding junction with the reservoir 604.At the junction where the aqueous fluid 608 and the second fluid 610meet, droplets can form based on factors such as the hydrodynamic forcesat the junction, flow rates of the two fluids 608, 610, fluidproperties, and certain geometric parameters (e.g., widths and heightsof the channel segments 602, expansion angle of the reservoir 604, etc.)of the channel structure 600, as described elsewhere herein. A pluralityof droplets can be collected in the reservoir 604 by continuouslyinjecting the aqueous fluid 608 from the plurality of channel segments602 through the plurality of junctions 606. Throughput may significantlyincrease with the substantially parallel channel configuration of thechannel structure 600. A channel structure may have as manysubstantially parallel channel segments as is practical and allowed forby the size of the reservoir. For example, the channel structure mayhave at least about 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70,80, 90, 100, 150, 200, 250, 300, 350, 400, 450, 500, 600, 700, 800, 900,1000, 1500, 5000 or more parallel or substantially parallel channelsegments. The plurality of channel segments may be substantially evenlyspaced apart, for example, around an edge or perimeter of the reservoir.Alternatively, the spacing of the plurality of channel segments may beuneven.

The reservoir 604 may have an expansion angle, α (not shown in FIG. 6 )at or near each channel junction. Each channel segment of the pluralityof channel segments 602 may have a width, w, and a height, h₀, at ornear the channel junction. The geometric parameters, w, h₀, and α, mayor may not be uniform for each of the channel segments in the pluralityof channel segments 602. For example, each channel segment may have thesame or different widths at or near its respective channel junction withthe reservoir 604. For example, each channel segment may have the sameor different height at or near its respective channel junction with thereservoir 604.

The reservoir 604 may have the same or different expansion angle at thedifferent channel junctions with the plurality of channel segments 602.For example, a circular reservoir (as shown in FIG. 6 ) may have aconical, dome-like, or hemispherical ceiling (e.g., top wall) to providethe same or substantially same expansion angle for each channel segments602 at or near the plurality of channel junctions 606. When thegeometric parameters are uniform, beneficially, resulting droplet sizemay be controlled to be uniform even with the increased throughput. Insome instances, when it is desirable to have a different distribution ofdroplet sizes, the geometric parameters for the plurality of channelsegments 602 may be varied accordingly.

In some instances, at least about 50% of the droplets generated can haveuniform size. In some instances, at least about 55%, 60%, 65%, 70%, 75%,80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or greater of the dropletsgenerated can have uniform size. Alternatively, less than about 50% ofthe droplets generated can have uniform size. The beads and/orbiological particle injected into the droplets may or may not haveuniform size.

FIG. 7A shows a cross-section view of another example of a microfluidicchannel structure with a geometric feature for controlled partitioning.A channel structure 700 can include a channel segment 702 communicatingat a channel junction 706 (or intersection) with a reservoir 704. Insome instances, the channel structure 700 and one or more of itscomponents can correspond to the channel structure 100 and one or moreof its components. FIG. 7B shows a perspective view of the channelstructure 700 of FIG. 7A.

An aqueous fluid 712 comprising a plurality of particles 716 may betransported along the channel segment 702 into the junction 706 to meeta second fluid 714 (e.g., oil, etc.) that is immiscible with the aqueousfluid 712 in the reservoir 704 to create droplets 720 of the aqueousfluid 712 flowing into the reservoir 704. At the junction 706 where theaqueous fluid 712 and the second fluid 714 meet, droplets can form basedon factors such as the hydrodynamic forces at the junction 706, relativeflow rates of the two fluids 712, 714, fluid properties, and certaingeometric parameters (e.g., Δh, etc.) of the channel structure 700. Aplurality of droplets can be collected in the reservoir 704 bycontinuously injecting the aqueous fluid 712 from the channel segment702 at the junction 706.

A discrete droplet generated may comprise one or more particles of theplurality of particles 716. As described elsewhere herein, a particlemay be any particle, such as a bead, cell bead, gel bead, biologicalparticle, macromolecular constituents of biological particle, or otherparticles. Alternatively, a discrete droplet generated may not includeany particles.

In some instances, the aqueous fluid 712 can have a substantiallyuniform concentration or frequency of particles 716. As describedelsewhere herein (e.g., with reference to FIG. 4 ), the particles 716(e.g., beads) can be introduced into the channel segment 702 from aseparate channel (not shown in FIG. 7 ). The frequency of particles 716in the channel segment 702 may be controlled by controlling thefrequency in which the particles 716 are introduced into the channelsegment 702 and/or the relative flow rates of the fluids in the channelsegment 702 and the separate channel. In some instances, the particles716 can be introduced into the channel segment 702 from a plurality ofdifferent channels, and the frequency controlled accordingly. In someinstances, different particles may be introduced via separate channels.For example, a first separate channel can introduce beads and a secondseparate channel can introduce biological particles into the channelsegment 702. The first separate channel introducing the beads may beupstream or downstream of the second separate channel introducing thebiological particles.

In some instances, the second fluid 714 may not be subjected to and/ordirected to any flow in or out of the reservoir 704. For example, thesecond fluid 714 may be substantially stationary in the reservoir 704.In some instances, the second fluid 714 may be subjected to flow withinthe reservoir 704, but not in or out of the reservoir 704, such as viaapplication of pressure to the reservoir 704 and/or as affected by theincoming flow of the aqueous fluid 712 at the junction 706.Alternatively, the second fluid 714 may be subjected and/or directed toflow in or out of the reservoir 704. For example, the reservoir 704 canbe a channel directing the second fluid 714 from upstream to downstream,transporting the generated droplets.

The channel structure 700 at or near the junction 706 may have certaingeometric features that at least partly determine the sizes and/orshapes of the droplets formed by the channel structure 700. The channelsegment 702 can have a first cross-section height, h₁, and the reservoir704 can have a second cross-section height, h₂. The first cross-sectionheight, h₁, and the second cross-section height, h₂, may be different,such that at the junction 706, there is a height difference of Δh. Thesecond cross-section height, h₂, may be greater than the firstcross-section height, h₁. In some instances, the reservoir maythereafter gradually increase in cross-section height, for example, themore distant it is from the junction 706. In some instances, thecross-section height of the reservoir may increase in accordance withexpansion angle, β, at or near the junction 706. The height difference,Δh, and/or expansion angle, β, can allow the tongue (portion of theaqueous fluid 712 leaving channel segment 702 at junction 706 andentering the reservoir 704 before droplet formation) to increase indepth and facilitate decrease in curvature of the intermediately formeddroplet. For example, droplet size may decrease with increasing heightdifference and/or increasing expansion angle.

The height difference, Δh, can be at least about 1 µm. Alternatively,the height difference can be at least about 1, 2, 3, 4, 5, 6, 7, 8, 9,10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, 50, 60,70, 80, 90, 100, 200, 300, 400, 500 µm or more. Alternatively, theheight difference can be at most about 500, 400, 300, 200, 100, 90, 80,70, 60, 50, 45, 40, 35, 30, 25, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11,10, 9, 8, 7, 6, 5, 4, 3, 2, 1 µm or less. In some instances, theexpansion angle, β, may be between a range of from about 0.5° to about4°, from about 0.1° to about 10°, or from about 0° to about 90°. Forexample, the expansion angle can be at least about 0.01°, 0.1°, 0.2°,0.3°, 0.4°, 0.5°, 0.6°, 0.7°, 0.8°, 0.9°, 1°, 2°, 3°, 4°, 5°, 6°, 7°,8°, 9°, 10°, 15°, 20°, 25°, 30°, 35°, 40°, 45°, 50°, 55°, 60°, 65°, 70°,75°, 80°, 85°, or higher. In some instances, the expansion angle can beat most about 89°, 88°, 87°, 86°, 85°, 84°, 83°, 82°, 81°, 80°, 75°,70°, 65°, 60°, 55°, 50°, 45°, 40°, 35°, 30°, 25°, 20°, 15°, 10°, 9°, 8°,7°, 6°, 5°, 4°, 3°, 2°, 1°, 0.1°, 0.01°, or less.

In some instances, the flow rate of the aqueous fluid 712 entering thejunction 706 can be between about 0.04 microliters (µL)/minute (min) andabout 40 µL/min. In some instances, the flow rate of the aqueous fluid712 entering the junction 706 can be between about 0.01 microliters(µL)/minute (min) and about 100 µL/min. Alternatively, the flow rate ofthe aqueous fluid 712 entering the junction 706 can be less than about0.01 µL/min. Alternatively, the flow rate of the aqueous fluid 712entering the junction 706 can be greater than about 40 µL/min, such as45 µL/min, 50 µL/min, 55 µL/min, 60 µL/min, 65 µL/min, 70 µL/min, 75µL/min, 80 µL/min, 85 µL/min, 90 µL/min, 95 µL/min, 100 µL/min, 110µL/min , 120 µL/min , 130 µL/min , 140 µL/min , 150 µL/min, or greater.At lower flow rates, such as flow rates of about less than or equal to10 microliters/minute, the droplet radius may not be dependent on theflow rate of the aqueous fluid 712 entering the junction 706. The secondfluid 714 may be stationary, or substantially stationary, in thereservoir 704. Alternatively, the second fluid 714 may be flowing, suchas at the above flow rates described for the aqueous fluid 712.

In some instances, at least about 50% of the droplets generated can haveuniform size. In some instances, at least about 55%, 60%, 65%, 70%, 75%,80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or greater of the dropletsgenerated can have uniform size. Alternatively, less than about 50% ofthe droplets generated can have uniform size.

While FIGS. 7A and 7B illustrate the height difference, Δh, being abruptat the junction 706 (e.g., a step increase), the height difference mayincrease gradually (e.g., from about 0 µm to a maximum heightdifference). Alternatively, the height difference may decrease gradually(e.g., taper) from a maximum height difference. A gradual increase ordecrease in height difference, as used herein, may refer to a continuousincremental increase or decrease in height difference, wherein an anglebetween any one differential segment of a height profile and animmediately adjacent differential segment of the height profile isgreater than 90°. For example, at the junction 706, a bottom wall of thechannel and a bottom wall of the reservoir can meet at an angle greaterthan 90°. Alternatively or in addition, a top wall (e.g., ceiling) ofthe channel and a top wall (e.g., ceiling) of the reservoir can meet anangle greater than 90°. A gradual increase or decrease may be linear ornon-linear (e.g., exponential, sinusoidal, etc.). Alternatively or inaddition, the height difference may variably increase and/or decreaselinearly or non-linearly. While FIGS. 7A and 7B illustrate the expandingreservoir cross-section height as linear (e.g., constant expansionangle, β), the cross-section height may expand non-linearly. Forexample, the reservoir may be defined at least partially by a dome-like(e.g., hemispherical) shape having variable expansion angles. Thecross-section height may expand in any shape.

The channel networks, e.g., as described above or elsewhere herein, canbe fluidically coupled to appropriate fluidic components. For example,the inlet channel segments are fluidly coupled to appropriate sources ofthe materials they are to deliver to a channel junction. These sourcesmay include any of a variety of different fluidic components, fromsimple reservoirs defined in or connected to a body structure of amicrofluidic device, to fluid conduits that deliver fluids fromoff-device sources, manifolds, fluid flow units (e.g., actuators, pumps,compressors) or the like. Likewise, the outlet channel segment (e.g.,channel segment 208, reservoir 604, etc.) may be fluidically coupled toa receiving vessel or conduit for the partitioned cells for subsequentprocessing. Again, this may be a reservoir defined in the body of amicrofluidic device, or it may be a fluidic conduit for delivering thepartitioned cells to a subsequent process operation, instrument orcomponent.

The methods and systems described herein may be used to greatly increasethe efficiency of single-cell applications and/or other applicationsreceiving droplet-based input. For example, following the sorting ofoccupied cells and/or appropriately-sized cells, subsequent operationsthat can be performed can include generation of amplification products,purification (e.g., via solid phase reversible immobilization (SPRI)),further processing (e.g., shearing, ligation of functional sequences,and subsequent amplification (e.g., via PCR)). These operations mayoccur in bulk (e.g., outside the partition). In the case where apartition is a droplet in an emulsion, the emulsion can be broken andthe contents of the droplet pooled for additional operations. Additionalreagents that may be co-partitioned along with the barcode bearing beadmay include oligonucleotides to block ribosomal RNA (rRNA) and nucleasesto digest genomic DNA from cells. Alternatively, rRNA removal agents maybe applied during additional processing operations. The configuration ofthe constructs generated by such a method can help minimize (or avoid)sequencing of the poly-T sequence during sequencing and/or sequence the5′ end of a polynucleotide sequence. The amplification products, forexample, first amplification products and/or second amplificationproducts, may be subject to sequencing for sequence analysis. In somecases, amplification may be performed using the Partial HairpinAmplification for Sequencing (PHASE) method.

A variety of applications require the evaluation of the presence andquantification of different biological particle or organism types withina population of biological particles, including, for example, microbiomeanalysis and characterization, environmental testing, food safetytesting, epidemiological analysis, e.g., in tracing contamination or thelike.

Computer Systems

The present disclosure provides computer systems that are programmed toimplement methods of the disclosure. FIG. 12 shows a computer system1201 that is programmed or otherwise configured to control amicrofluidics system (e.g., fluid flow) and perform sequencingapplications. The computer system 1201 can regulate various aspects ofthe present disclosure. The computer system 1201 can be an electronicdevice of a user or a computer system that is remotely located withrespect to the electronic device. The electronic device can be a mobileelectronic device.

The computer system 1201 includes a central processing unit (CPU, also“processor” and “computer processor” herein) 1205, which can be a singlecore or multi core processor, or a plurality of processors for parallelprocessing. The computer system 1201 also includes memory or memorylocation 1210 (e.g., random-access memory, read-only memory, flashmemory), electronic storage unit 1215 (e.g., hard disk), communicationinterface 1220 (e.g., network adapter) for communicating with one ormore other systems, and peripheral devices 1225, such as cache, othermemory, data storage and/or electronic display adapters. The memory1210, storage unit 1215, interface 1220 and peripheral devices 1225 arein communication with the CPU 1205 through a communication bus (solidlines), such as a motherboard. The storage unit 1215 can be a datastorage unit (or data repository) for storing data. The computer system1201 can be operatively coupled to a computer network (“network”) 1230with the aid of the communication interface 1220. The network 1230 canbe the Internet, an internet and/or extranet, or an intranet and/orextranet that is in communication with the Internet. The network 1230 insome cases is a telecommunication and/or data network. The network 1230can include one or more computer servers, which can enable distributedcomputing, such as cloud computing. The network 1230, in some cases withthe aid of the computer system 1201, can implement a peer-to-peernetwork, which may enable devices coupled to the computer system 1201 tobehave as a client or a server.

The CPU 1205 can execute a sequence of machine-readable instructions,which can be embodied in a program or software. The instructions may bestored in a memory location, such as the memory 1210. The instructionscan be directed to the CPU 1205, which can subsequently program orotherwise configure the CPU 1205 to implement methods of the presentdisclosure. Examples of operations performed by the CPU 1205 can includefetch, decode, execute, and writeback.

The CPU 1205 can be part of a circuit, such as an integrated circuit.One or more other components of the system 1201 can be included in thecircuit. In some cases, the circuit is an application specificintegrated circuit (ASIC).

The storage unit 1215 can store files, such as drivers, libraries andsaved programs. The storage unit 1215 can store user data, e.g., userpreferences and user programs. The computer system 1201 in some casescan include one or more additional data storage units that are externalto the computer system 1201, such as located on a remote server that isin communication with the computer system 1201 through an intranet orthe Internet.

The computer system 1201 can communicate with one or more remotecomputer systems through the network 1230. For instance, the computersystem 1201 can communicate with a remote computer system of a user(e.g., operator). Examples of remote computer systems include personalcomputers (e.g., portable PC), slate or tablet PC’s (e.g., Apple® iPad,Samsung® Galaxy Tab), telephones, Smart phones (e.g., Apple® iPhone,Android-enabled device, Blackberry®), or personal digital assistants.The user can access the computer system 1201 via the network 1230.

Methods as described herein can be implemented by way of machine (e.g.,computer processor) executable code stored on an electronic storagelocation of the computer system 1201, such as, for example, on thememory 1210 or electronic storage unit 1215. The machine executable ormachine readable code can be provided in the form of software. Duringuse, the code can be executed by the processor 1205. In some cases, thecode can be retrieved from the storage unit 1215 and stored on thememory 1210 for ready access by the processor 1205. In some situations,the electronic storage unit 1215 can be precluded, andmachine-executable instructions are stored on memory 1210.

The code can be pre-compiled and configured for use with a machinehaving a processor adapted to execute the code, or can be compiledduring runtime. The code can be supplied in a programming language thatcan be selected to enable the code to execute in a pre-compiled oras-compiled fashion.

Aspects of the systems and methods provided herein, such as the computersystem 1201, can be embodied in programming. Various aspects of thetechnology may be thought of as “products” or “articles of manufacture”typically in the form of machine (or processor) executable code and/orassociated data that is carried on or embodied in a type of machinereadable medium. Machine-executable code can be stored on an electronicstorage unit, such as memory (e.g., read-only memory, random-accessmemory, flash memory) or a hard disk. “Storage” type media can includeany or all of the tangible memory of the computers, processors or thelike, or associated modules thereof, such as various semiconductormemories, tape drives, disk drives and the like, which may providenon-transitory storage at any time for the software programming. All orportions of the software may at times be communicated through theInternet or various other telecommunication networks. Suchcommunications, for example, may enable loading of the software from onecomputer or processor into another, for example, from a managementserver or host computer into the computer platform of an applicationserver. Thus, another type of media that may bear the software elementsincludes optical, electrical and electromagnetic waves, such as usedacross physical interfaces between local devices, through wired andoptical landline networks and over various air-links. The physicalelements that carry such waves, such as wired or wireless links, opticallinks or the like, also may be considered as media bearing the software.As used herein, unless restricted to non-transitory, tangible “storage”media, terms such as computer or machine “readable medium” refer to anymedium that participates in providing instructions to a processor forexecution.

Hence, a machine readable medium, such as computer-executable code, maytake many forms, including but not limited to, a tangible storagemedium, a carrier wave medium or physical transmission medium.Non-volatile storage media include, for example, optical or magneticdisks, such as any of the storage devices in any computer(s) or thelike, such as may be used to implement the databases, etc. shown in thedrawings. Volatile storage media include dynamic memory, such as mainmemory of such a computer platform. Tangible transmission media includecoaxial cables; copper wire and fiber optics, including the wires thatcomprise a bus within a computer system. Carrier-wave transmission mediamay take the form of electric or electromagnetic signals, or acoustic orlight waves such as those generated during radio frequency (RF) andinfrared (IR) data communications. Common forms of computer-readablemedia therefore include for example: a floppy disk, a flexible disk,hard disk, magnetic tape, any other magnetic medium, a CD-ROM, DVD orDVD-ROM, any other optical medium, punch cards paper tape, any otherphysical storage medium with patterns of holes, a RAM, a ROM, a PROM andEPROM, a FLASH-EPROM, any other memory chip or cartridge, a carrier wavetransporting data or instructions, cables or links transporting such acarrier wave, or any other medium from which a computer may readprogramming code and/or data. Many of these forms of computer readablemedia may be involved in carrying one or more sequences of one or moreinstructions to a processor for execution.

The computer system 1201 can include or be in communication with anelectronic display 1235 that comprises a user interface (UI) 1240 forproviding, for example, results of sequencing analysis, etc. Examples ofUIs include, without limitation, a graphical user interface (GUI) andweb-based user interface.

Methods and systems of the present disclosure can be implemented by wayof one or more algorithms. An algorithm can be implemented by way ofsoftware upon execution by the central processing unit 1205. Thealgorithm can, for example, perform sequencing.

Devices, systems, compositions and methods of the present disclosure maybe used for various applications, such as, for example, processing asingle analyte (e.g., RNA, DNA, or protein) or multiple analytes (e.g.,DNA and RNA, DNA and protein, RNA and protein, or RNA, DNA and protein)from a single cell. For example, a biological particle (e.g., a cell orcell bead) is partitioned in a partition (e.g., droplet), and multipleanalytes from the biological particle are processed for subsequentprocessing. The multiple analytes may be from the single cell. This mayenable, for example, simultaneous proteomic, transcriptomic and genomicanalysis of the cell.

While preferred embodiments of the present invention have been shown anddescribed herein, it will be obvious to those skilled in the art thatsuch embodiments are provided by way of example only. It is not intendedthat the invention be limited by the specific examples provided withinthe specification. While the invention has been described with referenceto the aforementioned specification, the descriptions and illustrationsof the embodiments herein are not meant to be construed in a limitingsense. Numerous variations, changes, and substitutions will now occur tothose skilled in the art without departing from the invention.Furthermore, it shall be understood that all aspects of the inventionare not limited to the specific depictions, configurations or relativeproportions set forth herein which depend upon a variety of conditionsand variables. It should be understood that various alternatives to theembodiments of the invention described herein may be employed inpracticing the invention. It is therefore contemplated that theinvention shall also cover any such alternatives, modifications,variations or equivalents. It is intended that the following claimsdefine the scope of the invention and that methods and structures withinthe scope of these claims and their equivalents be covered thereby.

1-37. (canceled)
 38. A method for barcoding nucleic acid molecules of acell, comprising: (a) generating a cell bead comprising the cell,wherein the cell comprises a first nucleic acid molecule and a secondnucleic acid molecule, and wherein the first nucleic acid molecule andthe second nucleic acid molecule are different; (b) providing a firstsupport comprising a first nucleic acid barcode sequence and generatinga first barcoded nucleic acid molecule comprising (i) the first nucleicacid barcode sequence, and (ii) a sequence of the first nucleic acidmolecule; (c) providing a second support comprising a second nucleicacid barcode sequence that is different than the first nucleic acidbarcode sequence and generating: a second barcoded nucleic acid moleculecomprising (i) the second nucleic acid barcode sequence, and (ii) asequence of the second nucleic acid molecule, wherein the second nucleicacid barcode sequence is different from the first nucleic acid barcodesequence; and a third barcoded nucleic acid molecule comprising (i) thefirst nucleic acid barcode sequence; and (ii) the second nucleic acidbarcode sequence.
 39. The method of claim 38, further comprising (d)sequencing the first barcoded nucleic acid molecule or derivativethereof, the second barcoded nucleic acid molecule or derivativethereof, and the third barcoded nucleic acid molecule or derivativethereof.
 40. The method of claim 39, further comprising (e) usingsequencing reads generated in (d) to associate the first nucleic acidmolecule and the second nucleic acid molecule as having originated fromthe cell bead.
 41. The method of claim 38, wherein (a) comprises lysingthe cell.
 42. The method of claim 38, wherein (a) comprises attachingthe first nucleic acid molecule to the cell bead.
 43. The method ofclaim 42, wherein the first nucleic acid molecule is attached to thecell bead via a capture nucleic acid molecule configured to capture thefirst nucleic acid molecule.
 44. The method of claim 43, wherein thefirst nucleic acid molecule comprises messenger ribonucleic acid (mRNA).45. The method of claim 44, wherein the capture nucleic acid moleculecomprises a poly-T sequence.
 46. The method of claim 38, wherein thecell bead comprises an interpenetrating polymer network comprising afirst layer of polymer network and a second layer of polymer network.47. The method of claim 46, wherein the first layer of polymer networkdissociates by a first dissolution mechanism and the second layer ofpolymer network dissociates by a second dissolution mechanism, whereinthe first dissolution mechanism and the second dissolution mechanism aredifferent.
 48. The method of claim 47, wherein generating the cell beadcomprises applying the first dissolution mechanism, and whereinsubjecting the second partition to the second condition comprisesapplying the second dissolution mechanism, wherein the first dissolutionmechanism comprises a reduction or oxidation reaction.
 49. The method ofclaim 47, wherein the first dissolution mechanism releases the firstnucleic acid molecule from the cell bead without releasing the secondnucleic acid molecule from the cell bead, and wherein the seconddissolution mechanism releases the second nucleic acid molecule from thecell bead.
 50. The method of claim 46, wherein the first layer ofpolymer network comprises disulfide crosslinking bonds.
 51. The methodof claim 46, wherein the second layer of polymer network comprisesoxidizable crosslinking bonds.
 52. The method of claim 38, wherein thefirst nucleic acid molecule is a deoxyribonucleic acid molecule.
 53. Themethod of claim 38, wherein the first nucleic acid molecule is aribonucleic acid molecule.
 54. The method of claim 38, wherein the firstpartition or the second partition is a droplet.
 55. The method of claim38, wherein the first partition or the second partition is a well. 56.The method of claim 38, wherein the first bead or the second bead is agel bead.